WO1997011368A1 - Method for the analysis of allergen - Google Patents

Method for the analysis of allergen Download PDF

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Publication number
WO1997011368A1
WO1997011368A1 PCT/JP1996/002707 JP9602707W WO9711368A1 WO 1997011368 A1 WO1997011368 A1 WO 1997011368A1 JP 9602707 W JP9602707 W JP 9602707W WO 9711368 A1 WO9711368 A1 WO 9711368A1
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WO
WIPO (PCT)
Prior art keywords
allergen
antibody
labeled
wheat
extract
Prior art date
Application number
PCT/JP1996/002707
Other languages
French (fr)
Japanese (ja)
Inventor
Tadashi Matsunaga
Original Assignee
Asahi Denka Kogyo K.K.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Asahi Denka Kogyo K.K. filed Critical Asahi Denka Kogyo K.K.
Priority to DE69634558T priority Critical patent/DE69634558T2/en
Priority to EP96931261A priority patent/EP0794434B1/en
Publication of WO1997011368A1 publication Critical patent/WO1997011368A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials

Definitions

  • the present invention relates to a method for analyzing an allergen.
  • an allergen-labeled antibody complex is easily separated by ion exchange chromatography using a labeled antibody, a signal derived from the thus separated allergen-labeled antibody complex is measured, and the signal in the test sample is measured. Allergens can be analyzed immunologically. Background art
  • the most commonly used methods for measuring allergens in foods include, for example, an antibody labeled with a radioactive substance and indirect measurement of allergen antibody (anti-) ⁇ 'RAST (Radio -A 1 lergo Sorbent Test) method, ELISA (Enzyme-Linked Immuno Sorbent Assay) method that indirectly measures allergen antibody reaction using an enzyme-labeled antibody, or biological
  • PCA Passive Cutaneous Anaphy 1 axis
  • these methods are complicated to operate, and are not ready for analysis results. Takes time (several hours to one day). Also, when processing many samples, the operation is further complicated because the samples are divided into several groups and each is processed in a batch manner.
  • an object of the present invention is to provide a method which is simpler in operation than the conventional method in the analysis of allergens and is capable of rapidly quantifying the allergen.
  • Another object of the present invention is to provide another method which does not require cell preparation, unlike the above-mentioned electrochemical detection method.
  • a sample suspected of containing an allergen to be tested and a labeled antibody specific to the allergen to be tested are subjected to an antigen-antibody reaction between the allergen to be tested and the labeled antibody.
  • the method can be achieved by an allergen analysis method including a step of detecting a signal derived from the eluted test object allergen-labeled antibody complex.
  • FIG. 1 shows the structural formula of fluorescein isothiocyanate (FITC).
  • FIG. 2 shows a reaction formula between FITC and IgE antibody.
  • Fig. 4 is a diagram showing the separation of (DNP-BSA) -FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
  • Figure 5 shows cation exchange chromatography (DNP—BSA) -FI C
  • Figure 6 is a chart showing the separation of the TC-labeled antibody complex and unreacted labeled I g E antibody is a graph showing the reaction specificity of the FITC-labeled anti-DNP-I g E antibodies: 7 7 is a calibration curve showing the quantification of fluorescence intensity derived from (DNP-BSA) -FITC-labeled IgE antibody complex.
  • FIG. 8 is a chart showing separation of wheat allergen-alkaline phosphatase-labeled IgE antibody complex from unreacted alkaline phosphatase-labeled IgE antibody by cation exchange chromatography. .
  • FIG. 9 is a calibration curve showing the quantitativeness of the fluorescence intensity derived from the wheat allergen-alkaline phosphatase-labeled IgE antibody complex.
  • Figure 10 shows the results of cation exchange chromatography. This is a chart showing the separation of an alkaline phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody.
  • Figure 11 is a chart showing the separation of egg allergen-alkaline phosphatase labeled IgG antibody complex and unreacted alkaline phosphatase labeled IgG antibody by cation exchange chromatography. is there.
  • Fig. 12 is a chart showing the separation of rice allergen-alkaline phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. is there.
  • Fig. 13 shows the separation of wheat allergen-specific phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography.
  • Fig. 14 is a graph showing the separation of the descendant wheat allergen-alternative phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. It is.
  • Figure 15 shows the separation of the soy allergen-allelic phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. It is.
  • FIG. 16 is a calibration curve showing the quantification of the fluorescence intensity from the 3-alkoxyphosphatase-alkaline phosphatase-labeled IgE antibody complex:
  • FIG. 17 is a chart showing the separation of (-lactoglobulin) -one FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
  • FIG. 18 is a chart showing separation of an egg allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by positive ion exchange chromatography.
  • FIG. 19 is a chart showing separation of a rice allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
  • FIG. 20 is a chart showing separation of wheat allergen-FITC-labeled IgE antibody complex and unreacted F1 (labeled 1 gE antibody) by cation exchange chromatography.
  • FIG. 21 is a chart showing the separation of a wheat allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
  • FIG. 22 is a chart showing separation of a soy allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
  • FIG. 23 is a graph showing the quantification of the fluorescence intensity derived from the (DNP-BSA) -FITC-labeled IE antibody complex when continuously added to the column.
  • FIG. 24 is a chart showing separation of (DNP-BSA) —FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by anion exchange chromatography.
  • the sample that can be analyzed by the method of the present invention is not particularly limited as long as it is a sample suspected of containing an allergen.
  • foods that patients with allergic diseases may ingest, such as dairy products ( For example, milk, cheese, or yogurt), Eggs (eg, chicken eggs), cereals (eg, wheat or rice), legumes (eg, soy), vegetables, fruits, seafood, seafood, seaweed, meat, or processed foods thereof; pollen, eg, cedar, rice Or ragweed pollen; a medicament, eg, a vaccine or penicillin; animal hair, eg, dog or cat hair; a tick, eg, Dermatophagoides farinae or Dermatophagoides farinae; an insect, eg, Musselica; N. bacillus; fiber materials such as silk; or dust and dust in the room.
  • dairy products For example, milk, cheese, or yogurt
  • Eggs eg, chicken eggs
  • cereals eg, wheat or rice
  • legumes eg
  • a liquid sample can be used as it is as a sample. Alternatively, it can be used after dilution or extraction with an appropriate liquid (eg, water, saline, or buffer).
  • An appropriate liquid eg, water, saline, or buffer
  • a solid sample can be diluted or extracted with an appropriate liquid (eg, water, saline, or buffer) before use.
  • the allergen to be tested in the method of the present invention is not particularly limited as long as it is an allergen that causes an allergic disease.
  • proteins contained in various foods for example, lactoglobulin or ovomucoid can be exemplified.
  • proteins that are not isolated as allergens can be directly subjected to testing, for example, in the form of food extracts.
  • an antibody specific to the test allergen is prepared.
  • the class of the antibody specific to the test allergen is not particularly limited, for example, IgG, IgE, IgA, IgD, or IgM, preferably Ig G or IgE can be used. It is preferable to use IgG in terms of ease of antibody preparation, and it is preferable to use IgE in terms of sensitivity to allergic reactions.
  • An antibody specific to the test target allergen can be obtained by a known appropriate method. For example, after parenterally administering to an animal (particularly a mammal, for example, a rat, a mouse, or a heron) the parental apricot allergen as an immunogen and immunizing the animal, antiserum may be collected from the immunized animal. it can. Alternatively, a monoclonal antibody can be prepared by collecting spleen cells from the immunized animal and fusing the cells with myeloma cells. In addition, an antiserum is prepared by using a protein extract of an allergy-causing material (eg, food or pollen) as an immunogen. Forage pollen antibodies can also be prepared.
  • an allergy-causing material eg, food or pollen
  • the antibody thus obtained can be labeled by a known method.
  • the label that can be used in the method of the present invention is not particularly limited, and for example, a fluorescent compound, an enzyme, or a radioactive substance can be used.
  • a label In a fluorescence analysis using a fluorescent compound as a label, only light of an excitation wavelength is emitted to a sample, so that analysis can be performed in a short time. Luminescence analysis using enzymes has excellent sensitivity. In addition, analysis using radioactive materials is superior in analysis time and sensitivity. Therefore, in the method of the present invention, a label can be appropriately selected according to the purpose of analysis or the limit of quantification.
  • a fluorescent compound generally used in an immunological analysis method can be used as the fluorescent compound.
  • a compound that can be labeled by binding to an amino group of a protein is fluorescein thiosocyanate.
  • FITC tetramethylrhodamine isothiosinate
  • TRITC tetramethylrhodamine isothiosinate
  • dansyl mouth ride rhodamine isothiosinate
  • fluorescamine fluorescamine
  • benzonitoxazodiazole etc.
  • enzyme a luminescent enzyme generally used in an immunological analysis method can be used. These enzymes, substrates and chromogenic compounds are used in combination in a known manner. Enzymes may include, for example, alkaline phosphatase, 3,3-D-galactosidase, horseradish peroxidase, or catechol-10-methyltransferase.
  • radioactive substance a radioactive substance generally used in an immunological analysis method can be used.
  • the radioactive substance for example, can Rukoto cited I 1 2 5 or I c
  • Each of the above-mentioned labels can be bound to an antibody by a known appropriate method.
  • FITC is reacted with an antibody (protein) under alkaline conditions
  • the FITC isothiocyanate group and the antibody amino group (Mainly an amino group of a lysine group) react to form a stable dicarbamide bond. This reaction mechanism is shown in FIG.
  • the FITC and the antibody are mixed under alkaline conditions, preferably at pH 8 to 9, so that the molar ratio thereof is preferably 3 to 5: 1. Incubate at room temperature or below (preferably 15 to 25 ° C) for 2 hours or more (preferably 2 to 8 hours), or at about 4 overnight or more (preferably 8 to 20 hours). i.
  • Labeling of the antibody with the luminescent enzyme can be performed by a known and appropriate method. For example, a method in which the sugar chain hydroxyl group of the enzyme is reduced to form a Schiff base with the amino group of the antibody and the antibody is modified, or a disulfide bond in the antibody hinge is reduced to form the thiol group and the enzyme A method of bonding or cross-linking with an amino group of the above is known. At this time, it is preferable to use a cross-linking agent, because steric hindrance between the luminescent enzyme and the antibody is reduced or the binding molar ratio of the antibody and the luminescent enzyme is reduced. Powers that are not provided; N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP).
  • SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
  • labeling with a radioactive substance can be performed by a known appropriate method.
  • the chloramine T method can be mentioned. That is, the antibody is generally dissolved in a buffer of pH 7 and radioactive iodine is added to this solution: To this solution is added chloramine T (N-chloro-p-toluenesulfonamide) solution (500 ⁇ g / m 1) is added to give 10 mg of chloramine T per 1 mg of antibody, and radioiodinate for 10 minutes. Add sodium and sodium thiosulfate at 10 and 10 g / mg antibody, and stop) ', and remove unreacted radioactive iodine by dialysis
  • the labeled antibody is then separated from unreacted label and purified. Separation and refining
  • any appropriate method for example, dialysis or gel filtration column chromatography can be used.
  • the reaction solution containing the FITC-labeled antibody after the incubation is dialyzed against a buffer solution of near neutral to weakly alkaline (preferably pH 6 to 9) to obtain the unreacted solution.
  • the FITC can be removed and the FITC-labeled antibody can be purified, or the FITC-labeled antibody and unreacted FITC can be separated by gel filtration column chromatography. Separation by gel filtration chromatography-Separation of FITC (molecular weight 389.4) and antibody (IgE has a molecular weight of about 180,000, and IgG has a molecular weight of about 150,000) as a gel filtration carrier.
  • a crosslinked carrier such as dextran polymer, agarose, polyacrylamide, or a mixture of agarose and polyacrylamide having a pore size that can be used can be used.
  • the mobile phase may be a neutral or weakly alkaline buffer (preferably pH 7 to 8), such as a malonic acid buffer, a phosphate buffer, a carbonate buffer, a sodium citrate buffer, An acetate buffer, a sodium borate monohydroxide buffer, a glycine sodium monohydroxide buffer, a Tris-HCl buffer, or the like can be used.
  • the binding molar ratio between FITC and the antibody (FITCZ antibody) is not constant, and a mixture of labeled antibodies having various binding molar ratios is used. It is. Therefore, after the above-mentioned purification operation by dialysis or gel filtration chromatography, by treating with anion-exchange chromatography, fractions with a fixed binding molar ratio of FITC and antibody (FITCZ antibody) are fractionated. Is preferred.
  • the numerical value of the molar ratio is not particularly limited, it is preferably 1 to 2.
  • an anion exchange carrier for example, a weak anion exchange carrier using agarose beads is used.
  • An exchange carrier for example, a carrier having a getylaminoethyl (DEAE) group, a carrier having a cell mouth-sugel as a carrier, or a carrier having dextran as a carrier can be used.
  • the mobile phase may be a neutral or weakly alkaline (preferably pH 7 to 8) buffer, such as malonic acid buffer, phosphate buffer, carbonate buffer, sodium citrate monooxide buffer, An acetate buffer, a sodium borate monohydroxide buffer, a glycine sodium monohydroxide buffer, a Tris-HCl buffer, or the like can be used. Any suitable method can be used to separate the enzyme-labeled antibody from unreacted enzyme, and the radiolabeled antibody from unreacted radioactive material, such as dialysis or gel filtration columns. Chromatography and the like can be used.
  • a sample suspected of containing the test allergen is brought into contact with the labeled antibody.
  • Samples suspected of containing the test allergen include, as noted above, allergic substances-various substances that the patient may come into contact with, especially foods consumed by allergic patients, such as dairy products, eggs, or wheat Or processed foods thereof.
  • the contact is carried out, for example, in a weakly acidic to slightly alkaline (preferably pH 5 to 9) liquid.
  • a weakly acidic to slightly alkaline preferably pH 5 to 9
  • the protein is preferably solubilized with a salt solution of the above to prepare a sample.
  • liquid S3 ⁇ 4 fermented milk or the like it is preferable to adjust the pH to obtain a sample.
  • the contact between the sample and the labeled antibody is carried out under conditions that allow an antigen-antibody reaction between the test allergen contained in the sample and the labeled antibody.
  • conditions that allow an antigen-antibody reaction between the test allergen contained in the sample and the labeled antibody.
  • weakly acidic to weakly alkaline conditions preferably at pH 5 to 9
  • 37 or less preferably 25 to 37 ° C.
  • 20 minutes or more preferably 20 to 30 minutes
  • the obtained reaction solution is separated into an allergen-labeled antibody complex and unreacted labeled antibody by ion exchange chromatography.
  • the ion exchange chromatography that can be used in the method of the present invention is not particularly limited, but from the relationship between the isoelectric point of the labeled antibody protein used and the pH of the buffer as the mobile phase, Select an ion exchanger that can adsorb unreacted labeled antibodies. For example, when the labeled antibody to be used has an isoelectric point of 7 and a buffer having a pH of 5 as a mobile phase, a cation exchanger is used since the labeled antibody is positively charged.
  • the pH of the mobile phase buffer must be greater than 7, and the labeled antibody must be negatively charged.
  • enzyme labeling it is necessary to take into account the isoelectric point of the enzyme labeled with the antibody, and to make sure that the enzyme does not adsorb to the ion exchanger.
  • antibodies are weak to alkali, and in order to maintain antibody activity, it is preferable to use a cation exchanger whose mobile phase is acidic.
  • an optimal mobile phase buffer pH can be set from a combination of the isoelectric point of the labeled antibody and the exchangeable pH range of the ion exchanger.
  • conditions within which the unreacted labeled antibody adsorbs to the ion exchanger are selected so that the allergen-antibody complex does not adsorb to the ion exchanger. That is, when the antibody binds to the allergen to form a complex, the charge of the antibody changes, and the difference in electrostatic force between the antibody and the ion exchanger is reduced or the charge is reversed.
  • the adsorptivity between the complex and the ion exchanger is lower than the adsorptivity with the body, so that the labeled antibody can be adsorbed on the ion exchanger and only the complex can be eluted.
  • the cation exchange carrier that can be used in the method of the present invention is not particularly limited; for example, agarose beads, cellulose gel, or dextran, which is generally used in protein purification, is used as a carrier.
  • a cation exchange group such as a carboxymethyl group, a sulfomethyl group, a sulfoethyl group, or a phosphoric acid group can be used.
  • a malonic acid buffer, a phosphate buffer, a carbonate buffer, a sodium citrate monohydroxide buffer, or an acetate buffer is used. be able to.
  • the anion exchanger that can be used in the method of the present invention is not particularly limited.
  • an anion exchanger such as an aminoethyl group, a acetylaminoethyl group, or a guanidinoethyl group may be added to the carrier. Can be used.
  • the reaction solution obtained in the above contact step is treated by ion exchange chromatography, only the allergen-labeled antibody complex is eluted, and the unreacted labeled antibody is eluted by binding to the carrier in the ion exchange column. Therefore, the test target allergen-labeled antibody complex (that is, the test target allergen-fluorescence-labeled antibody complex, the test target allergen-emission enzyme complex, or the test target allergen-radioactive substance label) Antibody complex) and unreacted labeled antibody can be easily separated.
  • the test target allergen-labeled antibody complex that is, the test target allergen-fluorescence-labeled antibody complex, the test target allergen-emission enzyme complex, or the test target allergen-radioactive substance label
  • the unreacted labeled antibody bound to the carrier can be eluted with an aqueous solution having a salt concentration of 0.2 M or more (for example, 0.2 to 1 M), for example, an aqueous sodium chloride solution. Therefore, addition of the sample, elution of the test allergen-labeled antibody complex (target substance), elution of unreacted labeled antibody (non-target substance), and column equilibration by passing a weakly acidic buffer solution were performed. Since the cycle consisting of each operation can be repeatedly performed for one column one after another, the analyte can be continuously added to the column for analysis.
  • an alyte can be continuously added to the column for analysis.
  • the ion exchange capacity is within the ion exchange capacity of the column, elution of unreacted labeled antibody (non-target substance) and equilibration of the column by passing a weak acidic buffer are not necessarily performed. After eluting the test substance allergen-labeled antibody complex (target substance) from the previous reaction solution within the ion exchange capacity of, the next reaction solution can be continuously added. By doing so, each operation of dissolving the unreacted labeled antibody (non-target substance) and equilibrating the column by passing a weakly acidic buffer can be omitted, and the working efficiency is further improved.
  • the test allergen-label is obtained from the eluate containing the test allergen-labeled antibody complex eluted by the ion exchange chromatography.
  • a signal derived from the label in the antibody-antibody complex that is, the fluorescence from the fluorescent compound label, the luminescence derived from the color-forming enzyme, or the radioactivity derived from the radioactive substance enzyme is detected.
  • the calibration curve can be created by performing the same operation using a known concentration of the allergen. By comparing the measured value obtained for each sample with the above-mentioned calibration curve, the amount of allergen in the sample can be accurately quantified. It can also detect the presence of an allergen in a trial.
  • the detection or measurement of the fluorescence derived from the fluorescent compound label can be performed by any known method (preferably, an automatic analysis method) using any known device (for example, a fluorescence spectrophotometer). it can.
  • an appropriate substrate is added, and light having a wavelength corresponding to the substrate is applied to a known device (for example, a luminescence reader, that is, a luminescence analyzer). (Preferably an automatic analysis method).
  • alkaline phosphatase for example, fluorescein diphosphate, 4-methylpumbelliferyl phosphoric acid, 3_ (2, spiroadamantane) 14 -Methoxy 4- (3 "-Phosphoryloxy) phenyl-2-Doxetane (AMP PD), D-Luciferin-1 0-Phosphate (LUC P), or 0-Aminophthalylhydrazide 10-Phosphate (Luminol 10 -Phosphate, LUMP) can be used.
  • fluorescein diphosphate 4-methylpumbelliferyl phosphoric acid
  • 3D-galactosidase is used as a luminescent enzyme, for example, 3-(2 '-spiroadamantane) 1-4-methoxy 4- (3 "- ⁇ - ⁇ —galactopyranosi oxy) phenyl-1,2-dioxetane (AMPGD) or 0-amino-phthalylhydrazide-1,3-D-galactosido (Lumi Can be used.
  • AMPGD 3-(2 '-spiroadamantane) 1-4-methoxy 4- (3 "- ⁇ - ⁇ —galactopyranosi oxy) phenyl-1,2-dioxetane (AMPGD) or 0-amino-phthalylhydrazide-1,3-D-galactosido (Lumi Can be used.
  • AMPGD 0-amino-phthalylhydrazide-1,3-D-galactosido
  • horseradish peroxidase for example,
  • P-hydroxyphenylacetic acid, or p-hydroxyphenylpropionic acid can be used, and when catechol-10-methyltransferase is used as the luminescent enzyme, For example, 2- (3,4-dihydroxyphenyl) naphtho [1,2-d] thiazole (DNT) can be used.
  • a scintillation counter or the like can be used to measure the radioactivity derived from the radioactive substance label.
  • the step of detecting the signal derived from the allergen-labeled antibody complex can be performed in a batch system in which all the samples are sequentially performed.
  • the operations of the contacting step (1), the removing step (2), and the signal detecting step (3) are continuously performed, and then, the analyzing operation of the first sample is performed.
  • an analysis operation of the second sample is started at an appropriate interval, and the contacting step (1), the removing step (2), and the signal detecting step (3) are performed on the second sample.
  • the sample analysis operation is started one after another at appropriate intervals so as to continuously perform the above operations, and the contact step (1) and the removal step are performed for each of the following samples.
  • Example 1 1.8 ng 1 of FITC-labeled IgE antibody prepared in Example 1 (1) [in 0.01 M carbonate buffer (pH 9)] 1001 and DNP-BSA which is a model allergen (Dinitrophenylated bovine serum albumin) [0.011 ⁇ in carbonate buffer (H9)] 100 11 was mixed and the mixture was incubated at 371. At this time, the incubate (reaction) time was set to 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 60 minutes, and the concentration of the model allergen DNP-BSA was 0.6 mgZml. With 1.0 mgzml, 1.3 mgZml, or 2.
  • Example 1 the reaction specificity of the FITC-labeled anti-DNP-IgE antibody was examined.
  • Examples of the antigen test specimens were DNP—BSA, BSA (SIG used in Example 1 (1)).
  • MA powder
  • IgG SI GMA; liquid
  • HSA human blood '; blue albumin; Seikagaku Corporation; powder
  • the FITC-labeled IgE antibody and each antigen test sample were incubated at 371: 20 for 20 minutes. Thereafter, the fluorescence intensity of the antigen test sample-labeled antibody complex eluted with the malonate buffer used in Example 1 (3) was added to the ion exchange column used in Example 1 (3) and measured.
  • the concentration of D NP- BSA which is a model allergen
  • D NP- BSA which is a model allergen
  • a sample solution [0.05M malonic acid buffer (pH 5)] that was OmgZm or 5.0mgZm1 was prepared.
  • This DNP-BSA solution (100/1) was mixed with 1.8 ng Z1 of FITC-labeled IgE antibody solution (100/1) used in Example 1 (1) above. And incubated for 20 minutes.
  • Each reaction solution was treated in the same manner as in Example 1 (3), and the fluorescence intensity of the eluted allergen-labeled antibody complex was measured.
  • Figure 7 shows the results. When the allergen was in the range of 0 to 2 mg / m1, a linear correlation was shown.
  • Example 4 Analysis of wheat allergen by alkaline phosphatase-transfer agent Ichize labeled I g E antibodies
  • a rat immunized with the wheat allergen extract prepared in Example 4 (1) was used.
  • An anti-wheat allergen antiserum (2 ml) was obtained from the yeast.
  • the resulting precipitate was dialyzed for 48 hours in 2 liters of 0.01 M phosphate buffer (pH 8.0) for 48 hours: Then, the DEAE weakly basic The solution is added to an anion exchange column (5 ml; Itrapcolumn; manufactured by Pharmacia), and the phosphate buffer (pH 8) containing 0.15 M sodium chloride is used to prepare 0.01 M to 0.2 M phosphate. Elution was performed by applying a linear concentration gradient in the range described above, and an anti-wheat allergen-IgE antibody fraction was collected.
  • the obtained anti-wheat allergen-IgE antibody fraction was labeled with alkaline phosphatase by the following method.
  • SPDP-bound allelic phosphatase 1 ml of an anti-wheat allergen-IgE antibody fraction (1.1 mgZml) obtained by reducing the disulfide bond in the hinge region with dithiothreitol (DTT), After mixing with fat lm 1 and incubating at 4 ° C for 12 hours, the amino acid of alkaline phosphatase and anti-wheat allergen
  • Example 4 The alkaline phosphatase-labeled anti-wheat allergen-IgE ⁇ t form 501 prepared in Example 4 (2) and the wheat allergen extract (35 ⁇ g / ml) 50, prepared in Example 4 (1!) 1), incubated at 37 ° C for 20 minutes, and subjected to antigen-antibody reaction. (4) Fractionation of Allergen-Labeled Antibody Complex by Ion Exchange Chromatography After completion of the antigen-antibody reaction, this mixture 100, "1" was added to the cation exchange column used in Example 1 (3).
  • the mobile phase was eluted with a 50 mM malonate buffer (pH 5.0) at a flow rate of 1 m1 / min for 2 minutes, followed by a linear concentration gradient of NaC from 0 to 0.5 M.
  • the eluate was fractionated in 0.25 ml fractions as a fraction.
  • 3_ (2'-spiroadamantane) 14-methoxy-14- (3 "-phosphoryloxy) phenyl 1,2-dioxetane (AM PPD) 300 1 was used as a luminescent substrate in the analysis. After 15 minutes of incubation at 371C, the luminescence intensity was measured using Luminescence (Leader-1) (BLR 301; A10ka), and the results are shown in Figure 8. Then, only the allergen-labeled antibody complex (Peak A) eluted in the malonate buffer elution fraction and was passed through a NaCl aqueous solution with a linear concentration gradient of 0 to 0.5M.
  • Pak A allergen-labeled antibody complex
  • Alkaline phosphatase-labeled anti-wheat allergen-IgE antibody (Peak B) was eluted, that is, the unreacted alkaline phosphatase-labeled anti-wheat allergen-IgE antibody was unreacted.
  • the present invention is well-separated. It was possible to analyze the allergen by.
  • Example 4 (3) Using wheat allergen and alkaline phosphatase-labeled anti-wheat allergen-IgE antibody adjusted to a known amount in the same manner as in Example 4 (2) above, the procedure of Examples 4 (3) and (4) was repeated. The procedure was performed to test the quantitativeness. The results are shown in FIG. 9. c FIG. 9 shows the relative light intensity when the emission intensity when the entire sample was added to the column was 100%.
  • Example 6 Allergen measurement using alkaline phosphatase labeled IgG antibody
  • the rat was immunized with a milk allergen, one-lactoglobulin (SI GMA forest; powder; three times crystal preparation), and stakes,? 1 ml of antiglobulin antiserum 2 ml Obtained.
  • the obtained antiserum was added to a protein A column to obtain an IgG antibody fraction.
  • the same operation as in the above Example 4 (2) was carried out to obtain an anti-lipophosphatase-labeled anti- ,? One lactoglobulin-one IgG antibody was obtained.
  • the resulting anti-lipophosphatase-labeled anti-lactoglobulin-IgG antibody (20 ⁇ g / m 1: in 0.05M phosphate buffer) 50/1 and 3-lactogloprin (30 gZm l: 0 (In a 0.05M phosphate buffer) was incubated at room temperature for 20 minutes to carry out an antigen-antibody reaction.
  • rice, wheat, descendant wheat, and soybeans are each ground, and the ground material is homogenized by adding a 3% aqueous sodium chloride solution, and then centrifuged. Prepared. In addition, a diluted solution was prepared by diluting egg white with an equal amount of water. 50 ⁇ l of each extract or diluent (30 g protein / ml each) and the alkaline phosphatase-labeled anti- ,? One lactglobulin-IgG antibody (20 gZml) 501 was incubated at room temperature for 20 minutes to carry out an antigen-antibody reaction.
  • each of these reaction solutions 1001 was fractionated according to the method described in Example 4 (4), and the luminescence intensity was measured.
  • the results are shown in FIG. In FIG. 10, as the test sample,? The results when using a lactoglobulin solution (milk) are indicated by “ ⁇ ”, the results when using an egg white dilution are indicated by “ ⁇ ”, and the results when using a wheat extract are indicated by “A”.
  • the results using the descendant barley extract are shown as ⁇ X ''
  • the results using the soybean extract are shown as ⁇ * ''
  • the results using the rice extract are shown as ⁇ one ''.
  • the egg white dilution prepared in Example 6 (1) was used as an antigen, and the anti-egg allergen-IgG antibody fraction was prepared according to the method described in Example 6 (1). Using the IgG antibody fraction, an alkaline phosphatase-labeled anti-egg allergen-IgG antibody was prepared. The egg white dilution prepared in Example 6 (1), ⁇ -lactoglobulin The antigen-antibody reaction was performed with the solution, wheat extract, oat extract, soybean extract, or rice extract, and the antigen-antibody complex was analyzed.
  • results are shown in FIG. In Fig. 11, the results obtained when using the egg white diluent as the test sample are indicated by “ ⁇ ”, and the results obtained when a 3,3-lactoglobulin solution (milk) is used are indicated by “so-called”.
  • Use ⁇ for the result when using the liquid, X for the result using the bean extract, * for the result using the soybean extract, and use the rice extract when using the soybean extract The result of the above is indicated by “-”. Even when the anti-egg allergen-IgG was used, the egg allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed.
  • Example 6 (1) As an antigen instead of monolactoglobulin, an anti-rice allergen-IgG antibody fraction was prepared according to the method described in Example 6 (1). Using the IgG antibody fraction, an alkaline phosphatase-labeled anti-rice allergen-IgG antibody was prepared, and the egg white diluent prepared in Example 6 (1), a 3,3-lactoglobulin solution, An antigen-antibody reaction was performed with wheat extract, descendant barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex:
  • Example 6 (1) instead of lactoglobulin, the wheat extract prepared in Example 6 (1) was used as an antigen, and the anti-wheat allergen-IgG antibody fraction was used according to the method described in Example 6 (1).
  • the IgG white fraction was used to prepare an anti-wheat allergen-IgG antibody, which was used as an anti-wheat allergen, using the IgG antibody fraction.
  • the egg white diluent prepared in Example 6 (1) An antigen-antibody reaction was performed with a lactoglobulin solution, a wheat extract, a barley extract, a soybean extract, or a rice extract, and the antigen-antibody complex was analyzed.
  • the descendant wheat allergen-IgG antibody fraction was used according to the method described in Example 6 (1), using the descendant wheat extract prepared in Example 6 (1) as an antigen instead of lactoglobulin.
  • an alkaline phosphatase-labeled anti-allergen-IgG antibody was prepared, and the egg white diluent, lactoglobulin solution, and wheat prepared in Example 6 (1).
  • An antigen-antibody reaction was performed with the extract, barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex. The results are shown in FIG. In Fig.
  • egg white dilution is shown as “ ⁇ ” when the barley extract was used as the test sample, and as “solid” when the ⁇ -lactoglobulin solution (milk) was used as the test sample.
  • ⁇ A '' indicates the result when using the soybean extract
  • ⁇ X '' indicates the result when using the wheat extract
  • ⁇ * '' indicates the result when using the soybean extract
  • the rice extract was used. The result in each case is indicated by “one”.
  • the allergen-allergen-IgG the allergen-allergen-labeled antibody conjugate was well separated from the unreacted labeled antibody, and the allergen-labeled antibody conjugate could be analyzed.
  • test sample used was a lactoglobulin solution (milk), egg white diluent, wheat extract, soy extract, or rice extract, unreacted labeled anti-barley allergen-IgG Only the antibody was eluted.
  • Example 6 (1) The soybean extract prepared in Example 6 (1) was used as an antigen instead of lactoglobulin, and the anti-soy allergen-IgG antibody fraction was used according to the method described in Example 6 (1). Using the IgG antibody fraction, alkaline phosphatase-labeled anti-soy allergen-IgG antibody was prepared, and the egg white diluent and lactoglobulin solution prepared in Example 6 (1). An antigen-antibody reaction was performed with wheat extract, barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex.
  • results are shown in FIG. In Fig. 15, the results when soybean extract was used as the test sample are indicated by “ ⁇ ”, and the results when 3,3-lactoglobulin solution (milk) was used are indicated by “garden”.
  • ⁇ A '' indicates the result when using the extract
  • ⁇ X '' indicates the result when the wheat extract was used
  • ⁇ * '' indicates the result when the wheat extract was used
  • the results in each case are indicated by “-”. Even when the anti-soy allergen-IgG was used, the soy allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed.
  • test sample used was a lactoglobulin solution (milk), an egg A dilution, a wheat extract, a rose extract, or a rice extract, unreacted labeled anti-soy allergen-IG antibody Only eluted c
  • FIG. 16 shows the results.
  • FIG. 16 shows the relative luminescence intensity when the luminescence intensity of the whole sample added to the column is 100%.
  • Example 8 Analysis of food allergens by using 1 (: labeled 1 gE antibody
  • Each IgE antibody was obtained according to the method described in Example 4 (2) above, using the serum of each allergy patient to milk, egg white, rice, wheat, oats, or soybean. Using the obtained IgE antibodies of allergic patients, respective FITC-labeled IgE antibodies were obtained according to the method described in Example 1 (1).
  • Example 6 (1) As test samples, milk, egg white diluent, wheat extract, descendant barley extract, soybean extract, and rice extract prepared in Example 6 (1) were used.
  • the fluorescence intensity derived from the allergen-labeled antibody complex was measured according to the method described in Example 1 (3).
  • Fig. 17 shows the results obtained using FITC-labeled IgE antibodies prepared from IgE antibodies derived from milk allergy patients.
  • the symbols shown in FIG. 17 have the same meanings as the symbols shown in FIG.
  • the fluorescence from the allergen-labeled antibody complex is analyzed only for milk. it can, c the patient was able to more determined ⁇ Les Energy with respect to milk
  • FIGS. 18 to 22 have the same meanings as those shown in FIGS. 11 to 15, respectively.
  • FITC-labeled anti-DNP-IgE antibody of 1.8 ng z1 prepared in Example 1 (1) 100/1, 0.6 mg / ml, 1.0 mg / m1, 1. 3 mg / ml or 2. O mg of dinitrophenylated bovine serum albumin (DNP-BSA) 1001 was mixed with each other and incubated at 37 for 20 minutes. At this time, as a control test (blank), the same operation was performed using water instead of the DNP-BSA aqueous solution. The reaction mixture 100/1 was added every 20 minutes to the cation exchange column used in Example 1 (3), which had been equilibrated with 50 mM malonic acid buffer solution (pH 5.0). did.
  • DNP-BSA dinitrophenylated bovine serum albumin
  • the column was not washed with salt water at all between the sample addition and the next sample addition.
  • the addition was performed four times at each concentration of DNP-BSA.
  • a 50 mM malonic acid buffer solution pH 5.0
  • the eluate was fractionated in 0.25 ml fractions, and the fluorescence intensity of the eluate was measured with a fluorescence spectrophotometer (F-1200, [:] manufactured by Tachi Seisakusho).
  • the fluorescence intensity derived from the eluted antigen-antibody complex is shown in FIG. In Fig. 23, the point at which each reaction solution was added is indicated by “ ⁇ ”, and the addition of the reaction solution mixed with 2.
  • Omg / m1 of DNP-BSA was indicated by “A”, and the time of addition was 1.3 m.
  • the addition of the reaction mixture mixed with gZm1 DNP-BSA was performed with “B”, and the addition of the reaction mixture mixed with 1.0 mg / m1 DNP-BSA was added with “C” at 0.6 mg / m
  • the addition of the reaction mixture mixed with 1 DNP-BSA is indicated by “D”, and the addition of the reaction mixture mixed with water is indicated by “E”.
  • Example 1 (1) The same operation as in Example 1 (1) was performed, and the anti-DNP-IgE antibody was labeled with FITC. Subsequently, the same operation as in Example 1 (2) was performed, and FITC-labeled anti-DNP-IgE antibody (1. SngZ ⁇ l) 100 / l and DNP-BSA (model allergen)
  • allergens can be quickly quantified by a simple operation that has never been performed before.c

Abstract

A method for the analysis of an allergen comprising: (1) the step of contacting a specimen potentially containing the allergen to be examined with a labeled antibody being specific to the allergen under such conditions as to allow the allergen and the labeled antibody to undergo the antigen-antibody reaction; (2) the step of eliminating the labeled antibody which has failed to react with the allergen from the obtained reaction mixture by ion exchange chromatography; and (3) the step of detecting the signal originating in the allergen-antibody complex thus eluted. The method makes it possible to quickly assay the allergen by a more convenient procedure than those currently in use.

Description

明 細 書 アレルゲンの分析方法 技術分野  Description Allergen analysis method Technical field
本発明は、 アレルゲンの分析方法に関する。 本発明方法によれば、 標識抗体を 用い、 イオン交換クロマトグラフィーによりアレルゲン一標識抗体複合体を簡便 に分離し、 こうして分離したアレルゲン一標識抗体複合体由来の信号を計測し、 被検試料中のアレルゲンを免疫学的に分析 （アツセィ） することができる。 背景技術  The present invention relates to a method for analyzing an allergen. According to the method of the present invention, an allergen-labeled antibody complex is easily separated by ion exchange chromatography using a labeled antibody, a signal derived from the thus separated allergen-labeled antibody complex is measured, and the signal in the test sample is measured. Allergens can be analyzed immunologically. Background art
近年、 アレルギーは社会問題となっており、 その予防や治療に大きな関心が寄 せられている。 アレルギーの予防及び治療手段としては、 例えば、 食物アレルギ 一疾患患者用にアレルゲンを低減化した食品、 及びアレルゲンを含まない食品が 開発されている。 このような状況下で、 食品などの中に含まれるアレルゲンを分 析することは、 極めて重要な技術的課題となってきている。  In recent years, allergy has become a social problem, and there is great interest in its prevention and treatment. As means for preventing and treating allergies, for example, foods with reduced allergens for food allergic patients and foods without allergens have been developed. Under such circumstances, analyzing allergens in foods and the like has become a very important technical issue.
食品中に含まれるアレルゲンを測定する方法として従来から主に使用されてい るものとしては、 例えば、 放射性物質によって標識した抗体を用い、 アレルゲン 抗体反) ΐ'を間接的に測定する R A S T (R a d i o -A 1 l e r g o S o r b e n t T e s t ) 法、 酵素で標識した抗体を用いてアレルゲン抗体反応を間接 的に測定する EL I SA (E n z yme— L i n k e d I mmu n o S o r b e n t A s s a y) 法、 又は生体レベルでのアレルギー反応をラッ トの皮膚 反応で検知する PCA (P a s s i v e C u t a n e o u s A n a p h y 1 a x i s) 法等がある：： しかし、 これらの方法は、 操作も煩雑であり、 分析結果 が出るまでに時間を要する （数時間〜 1日） 。 また、 多くのサンプルを処理する 際には、 サンプルをいくつかのグループに分割し、 それぞれをバッチ式に処理す るため、 更に操作は煩雑なものになる。  The most commonly used methods for measuring allergens in foods include, for example, an antibody labeled with a radioactive substance and indirect measurement of allergen antibody (anti-) ΐ 'RAST (Radio -A 1 lergo Sorbent Test) method, ELISA (Enzyme-Linked Immuno Sorbent Assay) method that indirectly measures allergen antibody reaction using an enzyme-labeled antibody, or biological There are PCA (Passive Cutaneous Anaphy 1 axis) methods that detect allergic reactions at the skin level using rat skin reactions, etc .: However, these methods are complicated to operate, and are not ready for analysis results. Takes time (several hours to one day). Also, when processing many samples, the operation is further complicated because the samples are divided into several groups and each is processed in a batch manner.
このように、 食品などに含まれるアレルゲンを迅速且つ簡便に測定する適当な 方法は従来は存在せず、 アレルギー疾患患者用食品の製造時における品質管理が 大きな問題となっていた。 なお、 アレルギー反応のケミカルメデイエ一ターであ るセロトニンを指標として、 動物細胞を利用したアレルゲンの電気化学的検出方 法 （特開平 6— 288976号公報） も知られていた。 As described above, there has not been an appropriate method for quickly and easily measuring allergens contained in foods and the like, and quality control in the production of foods for patients with allergic diseases has not been performed. It was a big problem. In addition, a method of electrochemically detecting allergens using animal cells using serotonin, which is a chemical mediator of allergic reactions, as an index (Japanese Patent Application Laid-Open No. 6-288976) was also known.
従って、 本発明の目的は、 アレルゲンの分析において、 従来法と比較して操作 が簡便で、 しかも迅速にアレルゲンを定量することのできる方法を提供すること にある。  Accordingly, an object of the present invention is to provide a method which is simpler in operation than the conventional method in the analysis of allergens and is capable of rapidly quantifying the allergen.
また、 本発明の目的は、 前記の電気化学的検出方法とは異なり、 細胞の調製を 必要としない、 別の方法を提供することにもある。  Another object of the present invention is to provide another method which does not require cell preparation, unlike the above-mentioned electrochemical detection method.
本発明のその他の目的及び効果は、 以下の記載から明らかになろう。 発明の開示  Other objects and effects of the present invention will be apparent from the following description. Disclosure of the invention
前記の目的は、 本発明による、  The above object is according to the present invention,
( 1) 検査対象アレルゲンを含有する疑いのある試料と、 前記の検査対象アレル ゲンに特異的な標識抗体とを、 前記の検査衬象アレルゲンと前記標識抗体との抗 原抗体反応が可能な条件下で接触させる工程、  (1) A sample suspected of containing an allergen to be tested and a labeled antibody specific to the allergen to be tested are subjected to an antigen-antibody reaction between the allergen to be tested and the labeled antibody. Contacting underneath,
(2) 得られた反応液からイオン交換クロマトグラフィーにより、 検査対象ァレ ルゲンに未反応の標識抗体を除去する工程、 そして  (2) removing the unreacted labeled antibody from the allergen to be tested by ion exchange chromatography from the obtained reaction solution; and
(3) 溶出される検査対象ァレルゲン一標識抗体複合体由来の信号を検出するェ 程を含む、 アレルゲンの分析方法によって達成することができる。 図面の簡単な説明  (3) The method can be achieved by an allergen analysis method including a step of detecting a signal derived from the eluted test object allergen-labeled antibody complex. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 フルォレセインイソチオシァネート （F I TC) の構造式を示す。 図 2は、 F I TCと I gE抗体との反応式を示す。  FIG. 1 shows the structural formula of fluorescein isothiocyanate (FITC). FIG. 2 shows a reaction formula between FITC and IgE antibody.
図 3は、 （DNP— B SA) — F I TC標識 I g E抗体複合体量の経時変化を 示すグラフである c Figure 3, (DNP- B SA) - FI TC labeled I g E c is a graph showing the time course of antibody complex weight
図 4は、 陽イオン交換クロマトグラフィーによる、 （DNP— B SA) — F I TC標識 I gE抗体複合体と未反応の F I TC標識 I gE抗体との分離を示すチ 一トである  Fig. 4 is a diagram showing the separation of (DNP-BSA) -FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
図 5は、 陽イオン交換クロマトグラフィーによる、 （DNP— B SA) - F I T C標識抗体複合体と未反応の標識 I g E抗体との分離を示すチャー トである c 図 6は、 F I T C標識抗 D N P— I g E抗体の反応特異性を示すグラフである： 図 7は、 （D N P— B S A ) — F I T C標識 I g E抗体複合体に由来する蛍光 強度の定量性を示す検量線である。 Figure 5 shows cation exchange chromatography (DNP—BSA) -FI C Figure 6 is a chart showing the separation of the TC-labeled antibody complex and unreacted labeled I g E antibody is a graph showing the reaction specificity of the FITC-labeled anti-DNP-I g E antibodies: 7 7 is a calibration curve showing the quantification of fluorescence intensity derived from (DNP-BSA) -FITC-labeled IgE antibody complex.
図 8は、 陽イオン交換クロマトグラフィーによる、 小麦アレルゲン一アルカリ ホスファタ一ゼ標識 I g E抗体複合体と、 未反応のアルカリホスファタ一ゼ標識 I g E抗体との分離を示すチヤ一トである。  FIG. 8 is a chart showing separation of wheat allergen-alkaline phosphatase-labeled IgE antibody complex from unreacted alkaline phosphatase-labeled IgE antibody by cation exchange chromatography. .
図 9は、 小麦アレルゲン一アルカリホスファタ一ゼ標識 I g E抗体複合体に由 来する蛍光強度の定量性を示す検量線である。  FIG. 9 is a calibration curve showing the quantitativeness of the fluorescence intensity derived from the wheat allergen-alkaline phosphatase-labeled IgE antibody complex.
図 1 0は、 陽イオン交換クロマトグラフィーによる、 ？一ラク トグロプリ ン） 一アルカリホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファ ターゼ標識 I g G抗体との分離を示すチヤ一トである。  Figure 10 shows the results of cation exchange chromatography. This is a chart showing the separation of an alkaline phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody.
図 1 1 は、 陽イオン交換クロマトグラフィーによる、 卵アレルゲン一アルカリ ホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファタ一ゼ標識 I g G抗体との分離を示すチヤ一トである。  Figure 11 is a chart showing the separation of egg allergen-alkaline phosphatase labeled IgG antibody complex and unreacted alkaline phosphatase labeled IgG antibody by cation exchange chromatography. is there.
図 1 2は、 陽ィオン交換クロマトグラフィ一による、 米アレルゲン一アルカリ ホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファタ一ゼ標識 I g G抗体との分離を示すチヤ一トである。  Fig. 12 is a chart showing the separation of rice allergen-alkaline phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. is there.
図 1 3は、 陽イオン交換クロマトグラフィーによる、 小麦アレルゲン一アル力 リホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファタ一ゼ標 識 I g G抗体との分離を示すチヤ一トである c Fig. 13 shows the separation of wheat allergen-specific phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. Is c
図 1 4は、 陽イオン交換クロマトグラフィ一による、 裔麦アレルゲン一アル力 リホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファタ一ゼ標 識 I g G抗体との分離を示すチャー トである。  Fig. 14 is a graph showing the separation of the descendant wheat allergen-alternative phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. It is.
図 1 5は、 陽イオン交換クロマトグラフィーによる、 大豆アレルゲン一アル力 リホスファタ一ゼ標識 I g G抗体複合体と、 未反応のアルカリホスファタ一ゼ標 識 I g G抗体との分離を示すチヤ一トである。  Figure 15 shows the separation of the soy allergen-allelic phosphatase-labeled IgG antibody complex from unreacted alkaline phosphatase-labeled IgG antibody by cation exchange chromatography. It is.
図 1 6は、 3—ラク トグロプリン） 一アルカリホスファタ一ゼ標識 I g E抗 体複合体に由来する蛍光強度の定量性を示す検量線である： 図 1 7は、 陽イオン交換クロマトグラフィ一による、 （ ーラク トグロプリン〉 一 F I TC標識 I gE抗体複合体と、 未反応の F I TC標識 I gE抗体との分離 を示すチヤ一トである。 Figure 16 is a calibration curve showing the quantification of the fluorescence intensity from the 3-alkoxyphosphatase-alkaline phosphatase-labeled IgE antibody complex: FIG. 17 is a chart showing the separation of (-lactoglobulin) -one FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
図 1 8は、 陽ィォン交換クロマトグラフィ一による、 卵アレルゲン一 F I TC 標識 I gE抗体複合体と、 未反応の F I TC標識 I gE抗体との分離を示すチヤ ―トである。  FIG. 18 is a chart showing separation of an egg allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by positive ion exchange chromatography.
図 1 9は、 陽イオン交換クロマトグラフィーによる、 米アレルゲン一 F I TC 標識 I gE抗体複合体と、 未反応の F I TC標識 I gE抗体との分離を示すチヤ ―トである。  FIG. 19 is a chart showing separation of a rice allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
図 20は、 陽イオン交換クロマトグラフィーによる、 小麦アレルゲン一 F I T C標識 I gE抗体複合体と、 未反応の F 1丁( 標識1 gE抗体との分離を示すチヤ ―トである。  FIG. 20 is a chart showing separation of wheat allergen-FITC-labeled IgE antibody complex and unreacted F1 (labeled 1 gE antibody) by cation exchange chromatography.
図 2 1は、 陽イオン交換クロマトグラフィ一による、 裔麦アレルゲン一 F I T C標識 I gE抗体複合体と、 未反応の F ITC標識 I gE抗体との分離を示すチヤ ―トである。  FIG. 21 is a chart showing the separation of a wheat allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
図 22は、 陽イオン交換クロマトグラフィーによる、 大豆アレルゲン一 F I T C標識 I gE抗体複合体と、 未反応の F ITC標識 I gE抗体との分離を示すチヤ — トである。  FIG. 22 is a chart showing separation of a soy allergen-FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by cation exchange chromatography.
図 23は、 カラムへ連続添加した場合の、 （DNP— B SA) — F I TC標識 I E抗体複合体に由来する蛍光強度の定量性を示すグラフである。  FIG. 23 is a graph showing the quantification of the fluorescence intensity derived from the (DNP-BSA) -FITC-labeled IE antibody complex when continuously added to the column.
図 24は、 陰イオン交換クロマトグラフィーによる、 （DNP— B SA) — F I TC標識 I gE抗体複合体と、 未反応の F I TC標識 I gE抗体との分離を示 すチャー トである。 発明を実施するための最良の形態  FIG. 24 is a chart showing separation of (DNP-BSA) —FITC-labeled IgE antibody complex from unreacted FITC-labeled IgE antibody by anion exchange chromatography. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本 ¾明を詳細に説明する  Hereinafter, the present invention will be described in detail.
本発明方法で分析することのできる試料は、 アレルゲンを含有する疑いのある 試料であれば特に限定されず、 例えば、 アレルギー疾患患者が摂取することのあ る殆ど全ての食品、 例えば、 乳製品 （例えば、 牛乳、 チーズ、 又はヨーグルト） 、 卵 （例えば、 鶏卵） 、 穀類 （例えば、 小麦、 又は米） 、 豆類 （例えば、 大豆） 、 野菜類、 果物類、 魚介類、 海草類、 肉類、 又はそれらの加工食品；花粉、 例えば、 スギ、 イネ又はブタクサの花粉；医薬品、 例えば、 ワクチン又はペニシリ ン ；動 物の毛、 例えば、 ィヌ又はネコの毛； ダニ、 例えば、 コナヒヨウダニ、 又はャケ ヒヨウヒダニ；昆虫、 例えば、 ュスリカ ；カビ、 例えば、 力ンジダ菌；繊維材料、 例えば、 絹；あるいは室内のほこりや塵等を挙げることができる。 特に、 食品の 分析に適している。 The sample that can be analyzed by the method of the present invention is not particularly limited as long as it is a sample suspected of containing an allergen. For example, almost all foods that patients with allergic diseases may ingest, such as dairy products ( For example, milk, cheese, or yogurt), Eggs (eg, chicken eggs), cereals (eg, wheat or rice), legumes (eg, soy), vegetables, fruits, seafood, seafood, seaweed, meat, or processed foods thereof; pollen, eg, cedar, rice Or ragweed pollen; a medicament, eg, a vaccine or penicillin; animal hair, eg, dog or cat hair; a tick, eg, Dermatophagoides farinae or Dermatophagoides farinae; an insect, eg, Musselica; N. bacillus; fiber materials such as silk; or dust and dust in the room. Particularly suitable for food analysis.
液状の試料は、 そのまま試料として用いることができる。 あるいは、 適当な液 体 （例えば、 水、 生理食塩水、 又は緩衝液） で希釈又は抽出して用いることがで きる。 固体の試料は、 適当な液体 （例えば、 水、 生理食塩水、 又は緩衝液） で希 釈又は抽出して用いることができる。  A liquid sample can be used as it is as a sample. Alternatively, it can be used after dilution or extraction with an appropriate liquid (eg, water, saline, or buffer). A solid sample can be diluted or extracted with an appropriate liquid (eg, water, saline, or buffer) before use.
本発明方法における検査対象アレルゲンは、 アレルギー疾患の原因となるァレ ルゲンであれば特に限定されない。 例えば、 各種食品に含まれるタンパク質、 例 えば、 ーラク トグロブリン又はオボムコイ ド等を挙げることができる。 また、 アレルゲンとして単離されていないタンパク質でも、 例えば、 食品抽出物の形態 で、 そのまま検査対象とすることができる。  The allergen to be tested in the method of the present invention is not particularly limited as long as it is an allergen that causes an allergic disease. For example, proteins contained in various foods, for example, lactoglobulin or ovomucoid can be exemplified. In addition, even proteins that are not isolated as allergens can be directly subjected to testing, for example, in the form of food extracts.
本発明方法では、 最初に、 検査対象アレルゲンに特異的な抗体を調製する。 検 査対象アレルゲンに特異的な抗体のクラスは、 特に限定されるものではなく、 例 えば、 I g G、 I g E、 I g A、 I g D、 又は I g Mなど、 好ましくは I g G又 は I g Eを使用することができる。 抗体調製の容易さの点では、 I g Gを使用す ることが好ましく、 アレルギー反応の鋭敏さの点では、 I g Eを用いることが好 ましい。  In the method of the present invention, first, an antibody specific to the test allergen is prepared. The class of the antibody specific to the test allergen is not particularly limited, for example, IgG, IgE, IgA, IgD, or IgM, preferably Ig G or IgE can be used. It is preferable to use IgG in terms of ease of antibody preparation, and it is preferable to use IgE in terms of sensitivity to allergic reactions.
検査衬象アレルゲンに特異的な抗体は、 公知の適当な方法により得ることがで きる。 例えば、 検杏対象アレルゲンを免疫原として非経口的に動物 （特に、 哺乳 類、 例えば、 ラッ ト、 マウス、 又はゥサギ） に投与して免疫した後、 免疫動物か ら抗血清を採取することができる。 あるいは、 前記の免疫動物から脾細胞を採取 し、 ミエローマ細胞と細胞融合させてモノクローナル抗体を調製することができ る。 また、 免疫原として、 アレルギー原因材料 （例えば、 食品、 又は花粉） のタ ンパク抽出物を使用して抗血清を調製し、 例えば、 抗小麦アレルゲン抗体ゃ抗ス ギ花粉抗体を調製することもできる。 An antibody specific to the test target allergen can be obtained by a known appropriate method. For example, after parenterally administering to an animal (particularly a mammal, for example, a rat, a mouse, or a heron) the parental apricot allergen as an immunogen and immunizing the animal, antiserum may be collected from the immunized animal. it can. Alternatively, a monoclonal antibody can be prepared by collecting spleen cells from the immunized animal and fusing the cells with myeloma cells. In addition, an antiserum is prepared by using a protein extract of an allergy-causing material (eg, food or pollen) as an immunogen. Forage pollen antibodies can also be prepared.
こう して得られた抗体を、 公知の方法で標識することができる。 本発明方法で 用いることのできる標識は、 特に限定されるものではなく、 例えば、 蛍光性化合 物、 酵素、 又は放射性物質を用いることができる  The antibody thus obtained can be labeled by a known method. The label that can be used in the method of the present invention is not particularly limited, and for example, a fluorescent compound, an enzyme, or a radioactive substance can be used.
標識として蛍光性化合物を用いる蛍光分析では、 試料に励起波長の光を放射す るだけであるので、 短時間で分析することができる。 酵素を用いる発光分析は、 感度が優れている。 また、 放射性物質を用いる分析は、 分析時間及び感度とも優 れている。 従って、 本発明方法では、 分析の目的や定量限界に応じて、 標識を適 宜選択することができる。  In a fluorescence analysis using a fluorescent compound as a label, only light of an excitation wavelength is emitted to a sample, so that analysis can be performed in a short time. Luminescence analysis using enzymes has excellent sensitivity. In addition, analysis using radioactive materials is superior in analysis time and sensitivity. Therefore, in the method of the present invention, a label can be appropriately selected according to the purpose of analysis or the limit of quantification.
本発明方法では、 蛍光性化合物として、 一般に免疫学的分析方法で使用されて いる蛍光性化合物を使用することができる。 例えば、 タンパク質のァミノ基に結 合して標識することのできる化合物として、 フルォレセィンィソチオシァネ一ト In the method of the present invention, a fluorescent compound generally used in an immunological analysis method can be used as the fluorescent compound. For example, a compound that can be labeled by binding to an amino group of a protein is fluorescein thiosocyanate.
(以下、 F I T Cと称することがある） 、 テ トラメチルローダミ ンイソチオシァ ネー ト （T M R I T C ) 、 ダンシルク口ライ ド、 ローダミ ンイソチオシァネー ト、 フルォレスカミ ン、 又はクロロニト口べンゾォキサジァゾ一ル等を挙げることが でき、 タンパク質のチオール基に結合して標識することのできる化合物として、(Hereinafter may be referred to as FITC), tetramethylrhodamine isothiosinate (TMRITC), dansyl mouth ride, rhodamine isothiosinate, fluorescamine, or benzonitoxazodiazole, etc. As a compound that can be labeled by binding to a thiol group of a protein,
(ョ一 ドアセチルアミ ノエチル） ナフチルァミ ンスルホン酸 （A E A N S ) 、 ジ フルォレセインシスチン、 フルォレセインマ一キユリ酢酸、 又は S—マ一キユリ — N—ダンシルシスティンを挙げることができる c (® one Doasechiruami aminoethyl) Nafuchiruami Nsuruhon acid (AEANS), di full O receptacle in cystine, Furuoreseinma one Kiyuri acetate, or S- Ma one Kiyuri - c which may be mentioned N- dansyl cis Tin
酵素としても、 一般に免疫学的分析方法で使用されている発光用酵素を使用す ることができる。 これらの酵素と基質と発色性化合物とを公知の方法で組合わせ て用いる。 酵素としては、 例えば、 アルカリホスファターゼ、 ，3— D—ガラク ト シダ一ゼ、 西洋ヮサビペルォキシダーゼ、 又はカテコール一 0—メチルトランス フェラ一ゼを挙げることができる：  As the enzyme, a luminescent enzyme generally used in an immunological analysis method can be used. These enzymes, substrates and chromogenic compounds are used in combination in a known manner. Enzymes may include, for example, alkaline phosphatase, 3,3-D-galactosidase, horseradish peroxidase, or catechol-10-methyltransferase.
放射性物質としても、 一般に免疫学的分析方法で使用されている放射性物質を 使用することができる。 放射性物質としては、 例えば、 I 1 2 5又は I を挙げ ることができる c As the radioactive substance, a radioactive substance generally used in an immunological analysis method can be used. The radioactive substance, for example, can Rukoto cited I 1 2 5 or I c
これらの蛍光性物質及び発光用酵素の中で、 安定で、 抗体活性を阻害せず、 標 識化が安易であり、 また溶解度も良好で、 分光学的な特性が有利 （最大吸収波長 と最大蛍光波長が離れている） 等の理由からフルォレセイ ンィソチオシァネ一ト (F I TC) 力5'特に優れている c F I TCの構造を図 1に示す。 Among these fluorescent substances and luminescent enzymes, they are stable, do not inhibit antibody activity, are easy to label, have good solubility, and have excellent spectroscopic properties (maximum absorption wavelength). And shown for reasons such as the maximum fluorescence wavelength is away) the structure of Furuoresei Nisochioshiane Ichito (FI TC) force 5 'is particularly excellent c FI TC in FIG.
前記の各標識は、 公知の適切な方法により抗体に結合させることができる 例えば、 F I TCは、 抗体 （タンパク質） とアルカリ性条件下で反応させると、 F I TCのイソチオシァネ一ト基と抗体のァミノ基 （主にリジン基のァミノ基） が反応し、 安定なジカルバミ ド結合を形成することができる。 この反応機構を図 2に示す。  Each of the above-mentioned labels can be bound to an antibody by a known appropriate method. For example, when FITC is reacted with an antibody (protein) under alkaline conditions, the FITC isothiocyanate group and the antibody amino group (Mainly an amino group of a lysine group) react to form a stable dicarbamide bond. This reaction mechanism is shown in FIG.
従って、 F I TCを蛍光標識として使用する場合は、 アルカリ性条件下、 好ま しくは p H 8〜 9において、 F I TC及び抗体をそのモル比カ好ましくは 3〜 5 ： 1となるように混合し、 室温以下 （好ましくは 1 5〜 25 °C) で 2時間以上 （好 ましくは 2〜8時間） 、 若しくは約 4でで一晩以上 （好ましくは 8〜 20時間） ィンキュベ一 トすればょレ i。  Therefore, when FITC is used as a fluorescent label, the FITC and the antibody are mixed under alkaline conditions, preferably at pH 8 to 9, so that the molar ratio thereof is preferably 3 to 5: 1. Incubate at room temperature or below (preferably 15 to 25 ° C) for 2 hours or more (preferably 2 to 8 hours), or at about 4 overnight or more (preferably 8 to 20 hours). i.
また、 発光用酵素の抗体への標識は、 公知の適切な方法により実施することが できる。 例えば、 酵素の糖鎖水酸基を還元し、 抗体のァミノ基との間でシッフ塩 基を形成し、 抗体を修飾する方法、 又は抗体ヒンジ部のジスルフィ ド結合を還元 し、 生成するチオール基と酵素のァミノ基との間を結合若しくは架橋させる方法 などが知られている。 この際に、 架橋剤を用いると、 発光用酵素と抗体との立体 障害が低減されたり、 抗体と発光用酵素の結合モル比の分散が小さくなるので、 好ましい,： 前記架橋剤は、 特に限定されるものではない力；、 N—サクシンイ ミジ ル一3— (2—ピリジルジチォ） プロピネート （S PDP) 等を挙げることがで きる。  Labeling of the antibody with the luminescent enzyme can be performed by a known and appropriate method. For example, a method in which the sugar chain hydroxyl group of the enzyme is reduced to form a Schiff base with the amino group of the antibody and the antibody is modified, or a disulfide bond in the antibody hinge is reduced to form the thiol group and the enzyme A method of bonding or cross-linking with an amino group of the above is known. At this time, it is preferable to use a cross-linking agent, because steric hindrance between the luminescent enzyme and the antibody is reduced or the binding molar ratio of the antibody and the luminescent enzyme is reduced. Powers that are not provided; N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP).
更に、 放射性物質による標識も、 公知の適切な方法により実施することができ る。 例えば、 クロラ ミ ン T法を挙げることができる。 すなわち、 一般的には抗体 を p H 7の緩衝液に溶解し、 これに放射性ヨウ素を加える： この溶液にクロラミ ン T ( N—クロ口一 p— トルエンスルホンアミ ド） 液 （500〃 g/m 1 ) を抗 体 1 m gあたりクロラ ミ ン T 1 0 m gとなるように添加し、 1 0分間、 放射性ョ ゥ素化する。 チォ硫酸ナ ト リ ウムを抗体 1 mgあたり 1 0 ,リ g加え、 反) ΐ'を停止 させ、 未反応の放射性ヨウ素は透析により除去する  Further, labeling with a radioactive substance can be performed by a known appropriate method. For example, the chloramine T method can be mentioned. That is, the antibody is generally dissolved in a buffer of pH 7 and radioactive iodine is added to this solution: To this solution is added chloramine T (N-chloro-p-toluenesulfonamide) solution (500 µg / m 1) is added to give 10 mg of chloramine T per 1 mg of antibody, and radioiodinate for 10 minutes. Add sodium and sodium thiosulfate at 10 and 10 g / mg antibody, and stop) ', and remove unreacted radioactive iodine by dialysis
次いで、 標識化した抗体を、 未反応の標識から分離し、 精製する。 分離及び精 製方法としては、 任意の適切な方法、 例えば、 透析、 又はゲル濾過カラムクロマ トグラフィ一等を用いることができる。 The labeled antibody is then separated from unreacted label and purified. Separation and refining As a production method, any appropriate method, for example, dialysis or gel filtration column chromatography can be used.
例えば、 F I TC標識抗体を精製する場合は、 インキュベート後の F I TC標 識抗体を含む反応液を中性付近ないし弱アルカリ性 〈好ましくは pH6〜9) の 緩衝液中で透析することにより未反応の F I TCを除去し、 F I TC標識抗体を 精製するか、 あるいは、 ゲル濾過カラムクロマトグラフィーにより F I TC標識 抗体と未反応の F I TCとを分離することができる。 ゲル濾過クロマトグラフィ —により分離する場合には、 ゲル濾過担体として、 F I TC (分子量 389. 4) と抗体 （ I g Eは分子量約 180， 000、 I gGは分子量約 150， 000) とを分離することができるポアサイズを有するデキストラン重合体、 ァガロース、 ポリアクリルアミ ド、 又はァガロースとポリアクリルアミ ドとの混合体等の架橋 担体を用いることができる。 また、 移動相としては、 中性又は弱アルカリ性 （好 ましくは pH7〜8) の緩衝液、 例えば、 マロン酸緩衝液、 リン酸緩衝液、 炭酸 緩衝液、 クェン酸一水酸化ナトリウム緩衝液、 酢酸緩衝液、 ホウ酸一水酸化ナト リウム緩衝液、 グリシン一水酸化ナトリウム緩衝液、 又はトリス塩酸緩衝液等を 用いることができる。 ゲル濾過クロマトグラフィーの溶出液について、 抗体由来 の 280 n mの紫外線吸収と F I TC由来の 520 n m蛍光強度とを測定し、 両 波長のピークが重なる部分を分取することにより、 F I TC標識抗体を精製する ことができる = For example, when purifying the FITC-labeled antibody, the reaction solution containing the FITC-labeled antibody after the incubation is dialyzed against a buffer solution of near neutral to weakly alkaline (preferably pH 6 to 9) to obtain the unreacted solution. The FITC can be removed and the FITC-labeled antibody can be purified, or the FITC-labeled antibody and unreacted FITC can be separated by gel filtration column chromatography. Separation by gel filtration chromatography-Separation of FITC (molecular weight 389.4) and antibody (IgE has a molecular weight of about 180,000, and IgG has a molecular weight of about 150,000) as a gel filtration carrier. A crosslinked carrier such as dextran polymer, agarose, polyacrylamide, or a mixture of agarose and polyacrylamide having a pore size that can be used can be used. The mobile phase may be a neutral or weakly alkaline buffer (preferably pH 7 to 8), such as a malonic acid buffer, a phosphate buffer, a carbonate buffer, a sodium citrate buffer, An acetate buffer, a sodium borate monohydroxide buffer, a glycine sodium monohydroxide buffer, a Tris-HCl buffer, or the like can be used. For the eluate of the gel filtration chromatography, the UV absorption at 280 nm derived from the antibody and the 520 nm fluorescence intensity derived from FITC were measured, and the portion where the peaks of both wavelengths overlap was collected to obtain the FITC-labeled antibody. Can be purified =
前記の透析又はゲル濾過クロマトグラフィ一による精製操作によって得られる F I TC標識抗体においては、 F I TCと抗体との結合モル比 （F I TCZ抗体） が一定ではなく、 各種の結合モル比の標識抗体の混合物である。 従って、 前記の 透析又はゲル濾過クロマトグラフィーによる精製操作の後で、 陰イオン交換ク口 マトグラフィ一で処理することにより、 F I T Cと抗体の結合モル比 （F I TC Z抗体） が一定のものを分画するのが好ましい。 結合モル比が一定の標識抗体を 用いると、 結合モル比が種々の混合物である標識抗体を Wいる場合と比較して、 より信頼性の高いデータを得ることができる。 モル比の数値自体は特に限定され るものではないが、 1〜 2であることが好ましい。  In the FITC-labeled antibody obtained by the above-mentioned purification operation by dialysis or gel filtration chromatography, the binding molar ratio between FITC and the antibody (FITCZ antibody) is not constant, and a mixture of labeled antibodies having various binding molar ratios is used. It is. Therefore, after the above-mentioned purification operation by dialysis or gel filtration chromatography, by treating with anion-exchange chromatography, fractions with a fixed binding molar ratio of FITC and antibody (FITCZ antibody) are fractionated. Is preferred. When a labeled antibody having a constant binding molar ratio is used, more reliable data can be obtained as compared to the case where labeled antibodies having various binding molar ratios are used. Although the numerical value of the molar ratio is not particularly limited, it is preferably 1 to 2.
陰イオン交換担体としては、 例えば、 ァガロースビーズを担体とした弱陰ィ才 ン交換担体、 例えばジェチルアミノエチル （D E A E ) 基を有するもの、 セル口 —スゲルを担体とするもの、 又はデキストランを担体とするものを用いることが できる。 また、 移動相としては、 中性又は弱アルカリ性 （好ましくは p H 7〜 8 ) の緩衝液、 例えば、 マロン酸緩衝液、 リン酸緩衝液、 炭酸緩衝液、 クェン酸一水 酸化ナトリウム緩衝液、 酢酸緩衝液、 ホウ酸一水酸化ナトリウム緩衝液、 グリシ ン一水酸化ナトリウム緩衝液、 又はトリス塩酸緩衝液等を用いることができる。 酵素で標識された抗体と未反応の酵素との分離、 及び放射性物質で標識された 抗体と未反応の放射性物質との分離にも、 任意の適切な方法、 例えば、 透析、 又 はゲル濾過カラムクロマトグラフィー等を用いることができる。 As an anion exchange carrier, for example, a weak anion exchange carrier using agarose beads is used. An exchange carrier, for example, a carrier having a getylaminoethyl (DEAE) group, a carrier having a cell mouth-sugel as a carrier, or a carrier having dextran as a carrier can be used. The mobile phase may be a neutral or weakly alkaline (preferably pH 7 to 8) buffer, such as malonic acid buffer, phosphate buffer, carbonate buffer, sodium citrate monooxide buffer, An acetate buffer, a sodium borate monohydroxide buffer, a glycine sodium monohydroxide buffer, a Tris-HCl buffer, or the like can be used. Any suitable method can be used to separate the enzyme-labeled antibody from unreacted enzyme, and the radiolabeled antibody from unreacted radioactive material, such as dialysis or gel filtration columns. Chromatography and the like can be used.
次に、 本発明方法では、 検査対象アレルゲンを含有する疑いのある試料と前記 の標識抗体とを接触させる。  Next, in the method of the present invention, a sample suspected of containing the test allergen is brought into contact with the labeled antibody.
検査対象アレルゲンを含有する疑いのある試料とは、 前記のとおり、 アレルギ —疾患患者が接触することのある各種の物質、 特にアレルギー疾患患者が摂取す る食品、 例えば、 乳製品、 卵、 若しくは小麦、 又はそれらの加工食品等である。 前記の接触は、 例えば、 弱酸性から弱アルカリ性 （好ましくは p H 5〜 9 ) の液 体中で実施するので、 例えば、 固体状の小麦等を試料として用いる場合には、 0 . 5 M以下の塩溶液でタンパク質を可溶化して試料とするのが好ましい。 液状の S¾ 酵乳等を試料として用いる場合には、 p Hを調節して試料とするのが好ましい。 試料と標識抗体との前記接触は、 試料中に含まれている検査対象ァレルゲンと 前記標識抗体との抗原抗体反応が可能な条件下で実施する。 例えば、 弱酸性から 弱アルカリ性条件下 （好ましくは p H 5〜 9 ) において、 3 7で以下 （好ましく は 2 5〜 3 7 °C ) で 2 0分間以上 （好ましくは 2 0〜 3 0分間） インキュべ一ト すればよい。 特には、 標識抗体の抗体や標識としての酵素が失活せず、 かつ、 標 識物質を遊離させない条件を選択する必要がある。  Samples suspected of containing the test allergen include, as noted above, allergic substances-various substances that the patient may come into contact with, especially foods consumed by allergic patients, such as dairy products, eggs, or wheat Or processed foods thereof. The contact is carried out, for example, in a weakly acidic to slightly alkaline (preferably pH 5 to 9) liquid. For example, when solid wheat or the like is used as a sample, 0.5 M or less is used. The protein is preferably solubilized with a salt solution of the above to prepare a sample. When liquid S¾ fermented milk or the like is used as a sample, it is preferable to adjust the pH to obtain a sample. The contact between the sample and the labeled antibody is carried out under conditions that allow an antigen-antibody reaction between the test allergen contained in the sample and the labeled antibody. For example, under weakly acidic to weakly alkaline conditions (preferably at pH 5 to 9), at 37 or less (preferably 25 to 37 ° C.) for 20 minutes or more (preferably 20 to 30 minutes) Incubation is sufficient. In particular, it is necessary to select conditions under which the antibody of the labeled antibody and the enzyme as the label are not inactivated and the labeling substance is not released.
例えば、 精製した F I T C標識抗体を使用する場合には、 抗体が失活しないよ うに、 F I T C標識抗体と試料との混合液を 3 7 I：以下 （好ましくは 2 5〜 3 7 で） で 2 0分間以上 （好ましくは 2 0〜 3 0分間） インキュベー トする = For example, when using a purified FITC-labeled antibody, the mixture of the FITC-labeled antibody and the sample should be mixed at 37 I: or less (preferably at 25 to 37) so that the antibody is not deactivated. Incubate for more than a minute (preferably 20 to 30 minutes) =
こう して、 試料と標識抗体とを接触 （例えば、 インキュベート） させると、 試 料中に検査対象ァレルゲンが含まれている場合には、 そのアレルゲンの存在量に 応じて、 反応液中にアレルゲン一標識抗体複合体が形成される。 In this way, when the sample and the labeled antibody are brought into contact (for example, incubated), if the test allergen is contained in the sample, the amount of the allergen present is reduced. Accordingly, an allergen-labeled antibody complex is formed in the reaction solution.
本発明方法では、 続いて、 得られた反応液をイオン交換クロマ トグラフ ィーに より、 アレルゲン一標識抗体複合体と、 未反応の標識抗体との分離を行う。 本発明方法に用いることのできるイオン交換クロマトグラフィ一は、 特に限定 されるものではないが、 使用する標識された抗体タンパク質の等電点と、 移動相 である緩衝液の p Hとの関係から、 未反応標識抗体が吸着することができるよう なイオン交換体を選択する。 例えば、 使用する標識された抗体の等電点が 7であ り、 移動相として p Hが 5の緩衝液を用いる場合、 標識された抗体はプラスに帯 電するので陽イオン交換体を用いる。 陰イオン交換体を用いる場合は、 移動相の 緩衝液の p Hを 7より大きく し、 標識された抗体をマイナスに帯電させる必要が ある。 また酵素標識の場合は、 特に抗体に標識された酵素の等電点を考慮し、 酵 素側がイオン交換体に吸着しないよう配慮する必要がある。 なお、 一般に抗体は アルカリに弱く、 抗体活性を保持させるためには、 移動相が酸性である陽イオン 交換体を用いることが好ましい。 更に、 標識された抗体の等電点とイオン交換体 の交換可能 p H範囲との組合せから最適な移動相の緩衝液 p Hを設定することも できる。 この場合、 緩衝液の p Hにより、 抗体の失活ゃ標識物質の失活、 標識物 質の抗体からの遊離、 及び/又はアレルゲン一抗体複合体の解離が起きない条件 を設定する必要がある。 更に、 未反応の標識抗体がイオン交換体に吸着する条件 の範囲において、 アレルゲン一抗体複合体がイオン交換体に吸着しない条件を選 定する。 すなわち、 抗体がアレルゲンと結合して複合体を形成すると、 抗体の電 荷が変化し、 イオン交換体との静電力の差異が小さくなるか、 又は電荷が逆転す るので、 標識抗体とイオン交換体との吸着性よりも、 複合体とイオン交換体との 吸着性の方が低くなり、 標識抗体をイオン交換体に吸着させ、 複合体のみを溶出 させることができる。  In the method of the present invention, subsequently, the obtained reaction solution is separated into an allergen-labeled antibody complex and unreacted labeled antibody by ion exchange chromatography. The ion exchange chromatography that can be used in the method of the present invention is not particularly limited, but from the relationship between the isoelectric point of the labeled antibody protein used and the pH of the buffer as the mobile phase, Select an ion exchanger that can adsorb unreacted labeled antibodies. For example, when the labeled antibody to be used has an isoelectric point of 7 and a buffer having a pH of 5 as a mobile phase, a cation exchanger is used since the labeled antibody is positively charged. If an anion exchanger is used, the pH of the mobile phase buffer must be greater than 7, and the labeled antibody must be negatively charged. In the case of enzyme labeling, it is necessary to take into account the isoelectric point of the enzyme labeled with the antibody, and to make sure that the enzyme does not adsorb to the ion exchanger. In general, antibodies are weak to alkali, and in order to maintain antibody activity, it is preferable to use a cation exchanger whose mobile phase is acidic. Furthermore, an optimal mobile phase buffer pH can be set from a combination of the isoelectric point of the labeled antibody and the exchangeable pH range of the ion exchanger. In this case, it is necessary to set conditions that do not cause inactivation of the antibody, inactivation of the labeling substance, release of the labeling substance from the antibody, and / or dissociation of the allergen-antibody complex by the pH of the buffer. . Furthermore, conditions within which the unreacted labeled antibody adsorbs to the ion exchanger are selected so that the allergen-antibody complex does not adsorb to the ion exchanger. That is, when the antibody binds to the allergen to form a complex, the charge of the antibody changes, and the difference in electrostatic force between the antibody and the ion exchanger is reduced or the charge is reversed. The adsorptivity between the complex and the ion exchanger is lower than the adsorptivity with the body, so that the labeled antibody can be adsorbed on the ion exchanger and only the complex can be eluted.
本発明方法において使用することのできる陽イオン交換担体は、 特に制限され るものではない力；、 例えば、 タンパク質精製の際に一般に利用されるァガロース ビーズ、 セルロースゲル、 又はデキス トランを担体とし、 これに各種官能基、 例 えば、 カルボキシメチル基、 スルホメチル基、 スルホェチル基、 又はリ ン酸基等 の陽イオン交換基を担持したものを用いることができる。 また移動相としては、 弱酸性、 好ましくは p H 4 . 5〜 6の緩衝液が好ましく、 例えば、 マロン酸緩衝 液、 リン酸緩衝液、 炭酸緩衝液、 クェン酸一水酸化ナトリウム緩衝液、 又は酢酸 緩衝液等を用いることができる。 The cation exchange carrier that can be used in the method of the present invention is not particularly limited; for example, agarose beads, cellulose gel, or dextran, which is generally used in protein purification, is used as a carrier. In addition, those carrying various functional groups, for example, a cation exchange group such as a carboxymethyl group, a sulfomethyl group, a sulfoethyl group, or a phosphoric acid group can be used. As the mobile phase, A buffer having a weak acidity, preferably pH 4.5 to 6, is preferable. For example, a malonic acid buffer, a phosphate buffer, a carbonate buffer, a sodium citrate monohydroxide buffer, or an acetate buffer is used. be able to.
本発明方法において使用することのできる陰イオン交換体も、 特に制限される ものではない力'、 例えば、 上記担体にアミノエチル基、 ジェチルアミノエチル基、 又はグァニジノエチル基のような陰ィォン交換基を担持したものを用いることが できる。  The anion exchanger that can be used in the method of the present invention is not particularly limited. For example, an anion exchanger such as an aminoethyl group, a acetylaminoethyl group, or a guanidinoethyl group may be added to the carrier. Can be used.
前記の接触工程で得られた反応液を、 イオン交換クロマトグラフィ一で処理す ると、 アレルゲン一標識抗体複合体のみが溶出され、 未反応標識抗体はイオン交 換カラム中の担体に結合して溶出しないので、 前記の反応液から、 検査対象ァレ ルゲン一標識抗体複合体 （すなわち、 検査対象アレルゲン—蛍光標識抗体複合体、 検査対象アレルゲン一発光用酵素複合体、 又は検査対象アレルゲン—放射性物質 標識抗体複合体） と、 未反応の標識抗体とを簡単に分離することができる。  When the reaction solution obtained in the above contact step is treated by ion exchange chromatography, only the allergen-labeled antibody complex is eluted, and the unreacted labeled antibody is eluted by binding to the carrier in the ion exchange column. Therefore, the test target allergen-labeled antibody complex (that is, the test target allergen-fluorescence-labeled antibody complex, the test target allergen-emission enzyme complex, or the test target allergen-radioactive substance label) Antibody complex) and unreacted labeled antibody can be easily separated.
担体に結合した未反応標識抗体は、 0 . 2 M以上 （例えば、 0 . 2 〜 1 M) の 塩濃度をもつ水溶液、 例えば、 塩化ナトリウム水溶液により溶出することができ る。 従って、 試料の添加、 検査対象アレルゲン一標識抗体複合体 （目的物） の溶 出、 未反応の標識抗体 （非目的物） の溶出、 及び弱酸性緩衝液の通液によるカラ ムの平衡化の各操作からなるサイクルを、 1つのカラムについて次々に繰り返し て実施することができるので、 測定対象物を連続的にカラムに添加し、 分析する ことができる。  The unreacted labeled antibody bound to the carrier can be eluted with an aqueous solution having a salt concentration of 0.2 M or more (for example, 0.2 to 1 M), for example, an aqueous sodium chloride solution. Therefore, addition of the sample, elution of the test allergen-labeled antibody complex (target substance), elution of unreacted labeled antibody (non-target substance), and column equilibration by passing a weakly acidic buffer solution were performed. Since the cycle consisting of each operation can be repeatedly performed for one column one after another, the analyte can be continuously added to the column for analysis.
また、 イオン交換カラムのイオン交換容量内であれば、 未反応の標識抗体 （非 目的物） の溶出、 及び弱酸性緩衝液の通液によるカラムの平衡化は必ずしも行う 必要はなく、 イオン交換カラムのイオン交換容量内で先の反応液から検査対象ァ レルゲン一標識抗体複合体 （目的物） の溶出を行った後、 次の反応液を連続的に 添加することができる。 このようにすると、 未反応の標識抗体 （非目的物） の溶 出、 及び弱酸性緩衝液の通液によるカラムの平衡化の各操作が省くことができ、 さらに作業効率がよい。  If the ion exchange capacity is within the ion exchange capacity of the column, elution of unreacted labeled antibody (non-target substance) and equilibration of the column by passing a weak acidic buffer are not necessarily performed. After eluting the test substance allergen-labeled antibody complex (target substance) from the previous reaction solution within the ion exchange capacity of, the next reaction solution can be continuously added. By doing so, each operation of dissolving the unreacted labeled antibody (non-target substance) and equilibrating the column by passing a weakly acidic buffer can be omitted, and the working efficiency is further improved.
本発明方法では、 前記のィォン交換クロマトグラフィ一によって溶出された検 査対象アレルゲン一標識抗体複合体を含む溶出液から、 検査対象ァレルゲンー標 識抗体複合体における標識に由来する信号、 すなわち、 蛍光性化合物標識からの の蛍光、 発色用酵素に由来する発光、 又は放射性物質酵素に由来する放射能を検 出する。 検量線は、 既知濃度のアレルゲンを用い、 同様の操作を行い、 作成する ことができる。 個々の試料に関して得られた測定値を、 前記の検量線と比較して、 前記試科におけるアレルゲン量を正確に定量することができる。 また、 試科中で のアレルゲンの存在を検出することもできる。 In the method of the present invention, the test allergen-label is obtained from the eluate containing the test allergen-labeled antibody complex eluted by the ion exchange chromatography. A signal derived from the label in the antibody-antibody complex, that is, the fluorescence from the fluorescent compound label, the luminescence derived from the color-forming enzyme, or the radioactivity derived from the radioactive substance enzyme is detected. The calibration curve can be created by performing the same operation using a known concentration of the allergen. By comparing the measured value obtained for each sample with the above-mentioned calibration curve, the amount of allergen in the sample can be accurately quantified. It can also detect the presence of an allergen in a trial.
蛍光性化合物標識に由来する蛍光の検出又は測定は、 任意の公知の装置 （例え ば、 蛍光分光光度計） を用いて、 任意の公知の方法 （好ましくは自動分析法） に よって実施することができる。  The detection or measurement of the fluorescence derived from the fluorescent compound label can be performed by any known method (preferably, an automatic analysis method) using any known device (for example, a fluorescence spectrophotometer). it can.
酵素標識に由来する発光の検出又は測定は、 適当な基質を添加し、 基質に対応 した波長の光を公知の装置 （例えば、 ルミネッセンスリーダ一、 すなわち発光分 析器） を用いて、 任意の公知の方法 （好ましくは自動分析法） によって実施する ことができる。 前記の基質としては、 発光用酵素としてアルカリホスファタ一ゼ を使用する場合には、 例えば、 フルォレセイン二リン酸、 4ーメチルゥンベリフエ リルリ ン酸、 3 _ (2， ースピロアダマンタン） 一 4ーメ トキシー 4— (3" 一ホスホリロキシ） フエニル— 2—ジォキセタン （AMP PD) 、 D—ルシ フェリ ン一 0—ホスフェート （LUC P) 、 又は 0—ァミノフタリルヒ ドラジド 一 0—ホスフェー ト （ルミ ノール一 0—ホスフェー ト、 LUMP) を使用するこ とができ、 発光用酵素として, 3— D—ガラク トシダーゼを使用する場合には、 例 えば、 3— ( 2 ' —スピロアダマンタン） 一 4—メ トキシ一 4— (3" - β -Ό —ガラク トピラノシ口キシ） フエニル一 1， 2—ジォキセタン （AMPGD) 、 又は 0—アミ ノフタリルヒ ドラジド一 ,3— D—ガラク トシド （ルミ ノール一 ,？ _D—ガラク トシド、 LUMG) を使用することができ、 発光用酵素として西洋 ヮサビペルォキシダ一ゼを使用する場合には、 例えば、 ホモバニリン酸、 チラミ ン、 p—クレゾ一ル、 p—ヒ ドロキシフエ二ル酢酸、 又は p—ヒ ドロキシフエ二 ルプロピオン酸 （HP PA) を使用することができ、 そして、 発光用酵素にカテ コール一 0—メチル トランスフェラ一ゼを用いる場合には、 例えば、 2— (3， 4ージヒ ドロキシフエニル） ナフト [ 1 , 2— d] チアゾール （DNT) を使用 することができる。 放射性物質標識に由来する放射能の測定には、 シンチレーシヨンカウンタ一等 を使用することができる。 For detection or measurement of luminescence derived from the enzyme label, an appropriate substrate is added, and light having a wavelength corresponding to the substrate is applied to a known device (for example, a luminescence reader, that is, a luminescence analyzer). (Preferably an automatic analysis method). When alkaline phosphatase is used as the luminescent enzyme as the substrate, for example, fluorescein diphosphate, 4-methylpumbelliferyl phosphoric acid, 3_ (2, spiroadamantane) 14 -Methoxy 4- (3 "-Phosphoryloxy) phenyl-2-Doxetane (AMP PD), D-Luciferin-1 0-Phosphate (LUC P), or 0-Aminophthalylhydrazide 10-Phosphate (Luminol 10 -Phosphate, LUMP) can be used. If 3D-galactosidase is used as a luminescent enzyme, for example, 3-(2 '-spiroadamantane) 1-4-methoxy 4- (3 "-β-Ό—galactopyranosi oxy) phenyl-1,2-dioxetane (AMPGD) or 0-amino-phthalylhydrazide-1,3-D-galactosido (Lumi Can be used. When using horseradish peroxidase as a luminescent enzyme, for example, homovanillic acid, tyramine, p-cresol can be used. , P-hydroxyphenylacetic acid, or p-hydroxyphenylpropionic acid (HP PA) can be used, and when catechol-10-methyltransferase is used as the luminescent enzyme, For example, 2- (3,4-dihydroxyphenyl) naphtho [1,2-d] thiazole (DNT) can be used. A scintillation counter or the like can be used to measure the radioactivity derived from the radioactive substance label.
複数の試料を処理する場合には、 例えば、  When processing multiple samples, for example,
(1 ) 試料と標識抗体との接触工程を、 検査すべき全て試料に関して実施し、 続 いて  (1) Perform the contacting step between the sample and the labeled antibody on all the samples to be tested.
(2) イオン交換クロマトグラフィーによる、 未反応標識抗体の除去工程を、 検 査すべき全て試料に関して実施し、 そして、 それらの試料について  (2) Perform the step of removing unreacted labeled antibody by ion exchange chromatography on all the samples to be tested, and perform the
(3) アレルゲン一標識抗体複合体由来の信号の検出工程を、 全試料に関して順 に実施する、 バッチ式で行うことができる。  (3) The step of detecting the signal derived from the allergen-labeled antibody complex can be performed in a batch system in which all the samples are sequentially performed.
あるいは、 第 1の試料について、 前記接触工程 （1 ) 、 前記除去工程 （2) 、 及び前記信号検出工程 （3) の操作を連続的に実施し、 続いて、 第 1の試料の分 析操作開始後に、 適切なインタ一バルをあけて第 2の試料の分析操作を開始し、 第 2の試料について前記接触工程 （1) 、 前記除去工程 （2) 、 及び前記信号検 出工程 （3) の操作を連続的に実施するように、 適当な間隔をあけて次々に試料 の分析操作を開始し、 以下の各試料について前記接触工程 （1 ) 、 前記除去工程 Alternatively, for the first sample, the operations of the contacting step (1), the removing step (2), and the signal detecting step (3) are continuously performed, and then, the analyzing operation of the first sample is performed. After the start, an analysis operation of the second sample is started at an appropriate interval, and the contacting step (1), the removing step (2), and the signal detecting step (3) are performed on the second sample. The sample analysis operation is started one after another at appropriate intervals so as to continuously perform the above operations, and the contact step (1) and the removal step are performed for each of the following samples.
(2) 、 及び前記信号検出工程 （3) の操作を連続的に実施することもできる c 連続的に行うと、 反応や測定を待つ時間が短縮され、 また、 アレルゲンの分析を 容易に自動化することができる。 実施例 (2) and the signal detection step (3) can be performed continuously. C If performed continuously, the waiting time for reaction and measurement can be reduced, and the analysis of allergens can be easily automated. be able to. Example
以下、 実施例により本発明を具体的に説明する力'、 これらは本発明を限定する ものではない c Hereinafter, the ability to specifically explain the present invention by way of Examples, which do not limit the present invention c
実施例 1 ： アレルゲン一標識抗体複合体の調製 Example 1: Preparation of allergen-labeled antibody complex
( 1 ) F I TC標識抗 DNP— I g E抗体の調製  (1) Preparation of F ITC-labeled anti-DNP-Ig E antibody
0. 51^炭酸緩衝液 （ロ^19) 1 5m l中に F I TC (フルォレセインイソチ オシァネート） 1 Omg及び抗 DNP (ジニトロフエニル） I gE抗体 （生化学 工業製；液状） 5 Omgを加え、 室温で 4時間インキュベートした。 この後、 1 5 m 1 を 0. 0 1 M炭酸緩衝液 （ p H 9 ) 3リ ッ トル中で 4 °Cで 72時間透析を 行うことにより、 未反応の F I TCを除去した。 (2) 試料との接触 0.51 ^ Carbonate buffer (b ^ 19) 1 Omg of FITC (fluorescein isothiocyanate) and 5 Omg of anti-DNP (dinitrophenyl) IgE antibody (manufactured by Seikagaku Corporation; liquid) in 15 ml Incubated at room temperature for 4 hours. After that, unreacted FITC was removed by dialysis of 15 ml in 3 liters of 0.01 M carbonate buffer (pH 9) at 4 ° C for 72 hours. (2) Contact with sample
実施例 1 ( 1) で調製した 1. 8 n g 1の F I TC標識 I g E抗体 〔0. 0 1 M炭酸緩衝液 （ p H 9 ) 中〕 1 00 1 と、 モデルアレルゲンである D N P 一 B S A (ジニトロフヱニル化牛血清アルブミ ン） 〔0. 0 11^炭酸緩衝液 （ H 9) 中〕 1 00〃 1 とを混合し、 371 でィンキュベ一トした。 この際、 ィン キュペート （反応） 時間を 1分間、 5分間、 10分間、 20分間、 30分間、 4 0分間、 又は 60分間とし、 またモデルアレルゲンである DNP— B S A濃度を 0. 6mgZm l、 1. 0mgzm l、 1. 3mgZm l、 又は 2. OmgZm 1 とし、 形成されるアレルゲン一標識抗体複合体量の経時変化を調べた。 結果を 図 3に示す。 図 3において、 口は DNP— BSA2mgZm 1の場合、 △は DN P— B SA 1. 3mgZm lの場合、 〇は D N P— B S A 1 m g 1の場合、 そして秦は DNP— B S A 0. 6 mgZm 1の場合である。 図 3から明らかなよ うに、 インキュベー ト （接触） 時間が 20分間以上になると、 それぞれの DNP 一 B S Aの濃度において蛍光強度が定常になることが分かる。  1.8 ng 1 of FITC-labeled IgE antibody prepared in Example 1 (1) [in 0.01 M carbonate buffer (pH 9)] 1001 and DNP-BSA which is a model allergen (Dinitrophenylated bovine serum albumin) [0.011 ^ in carbonate buffer (H9)] 100 11 was mixed and the mixture was incubated at 371. At this time, the incubate (reaction) time was set to 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 60 minutes, and the concentration of the model allergen DNP-BSA was 0.6 mgZml. With 1.0 mgzml, 1.3 mgZml, or 2. OmgZm1, the change over time in the amount of allergen-labeled antibody complex formed was examined. The results are shown in Figure 3. In Figure 3, the mouth is DNP-BSA2mgZm1, △ is DNP-BSA1.3mgZml, △ is DNP-BSA1mg1 and Hata is DNP-BSA0.6mgZm1. Is the case. As is clear from Fig. 3, when the incubation (contact) time is longer than 20 minutes, the fluorescence intensity becomes steady at each DNP-BSA concentration.
(3) イオン交換クロマトグラフィーによるアレルゲン—標識抗体複合体の分画 実施例 1 (2) で調製した反応液 （インキュべ一 ト時間 = 20分問； DNP 一 B S A濃度二 2 mgZm 1 ) 200," 1を、 陽イオン交換カラム （H I TRA P™S P-S e p h a r o s e F a s t F 1 o w；直径 = 1 6 X 25 mm； フアルマシア製） に添加した = 移動相としては、 50 m Mマロン酸緩衝液 ( p H 5. 0) を流速 I m l Zm i nで用いた。 また検出には、 蛍光分光光度計 （F - 2000 ； 日立製作所製） を用い、 F I TC由来の蛍光強度を測定した。 結果 を図 4に示す。 (3) Fractionation of allergen-labeled antibody complex by ion-exchange chromatography Reaction solution prepared in Example 1 (2) (incubation time = 20 minutes; DNP-BSA concentration-2 mgZm 1) 200, "1, cation exchange column as the (HI TRA P ™ S PS epharose F ast F 1 ow;; diameter = 1 6 X 25 mm Pharmacia Ltd.) to was added = mobile phase, 50 m M malonic acid buffer ( pH 5.0) was used at a flow rate of I ml Zmin, and the fluorescence intensity derived from FITC was measured using a fluorescence spectrophotometer (F-2000; manufactured by Hitachi, Ltd.). See Figure 4.
図 4から明らかなように、 アレルゲン一標識抗体複合体のみがマ口ン g緩衝液 溶出画分で溶出した アレルゲン一標識抗体複合体の溶出後、 0. 5 Mの N a C 1水溶液を通液したところ、 図 5に示すように、 未反応標識 I gE抗体が溶出さ れた。  As is evident from Fig. 4, only the allergen-labeled antibody complex was eluted in the eluate fraction of the mouse g buffer.After elution of the allergen-labeled antibody complex, a 0.5 M NaCl aqueous solution was passed through As a result, unreacted labeled IgE antibody was eluted as shown in FIG.
実施例 2 ： F I T C標識抗 DN P— I g E抗体の反応特異性 Example 2: Reaction specificity of FITC-labeled anti-DNP-IgE antibody
本実施例では、 F I TC標識抗 DNP— I g E抗体の反応特異性を調べた。 抗 原試験体としては、 実施例 1 ( 1 ) で使用した DNP— B S A、 B SA (S I G MA社製；粉末） 、 I g G (S I GMA社製；液状） 、 H S A (ヒ ト血';青アルブ ミ ン ；生化学工業製；粉末） を用い、 実施例 1 ( 1 ) で調製した F I TC標識 I g E抗体と各抗原試験体とを 3 71：で 2 0分間インキュベートした。 その後、 実 施例 1 ( 3 ) で使用したイオン交換カラムに添加し、 実施例 1 ( 3 ) で使用した マロン酸緩衝液で溶出される抗原試験体一標識抗体複合体の蛍光強度を測定した： 対照試験 （ブランク） は、 抗原試験体の代わりにリン酸緩衝生理食塩水を使用し て実施した。 その結果を図 6に示す。 B SA、 I g G, 及び H SAでは蛍光強度 の増加は認められないのに対し、 DNP— B S Aでは蛍光強度の増加が確認され た。 従って、 本発明方法により、 アレルゲンの選択的な測定が可能であることが 示された。 In this example, the reaction specificity of the FITC-labeled anti-DNP-IgE antibody was examined. Examples of the antigen test specimens were DNP—BSA, BSA (SIG used in Example 1 (1)). MA (powder), IgG (SI GMA; liquid), HSA (human blood '; blue albumin; Seikagaku Corporation; powder) were prepared in Example 1 (1). The FITC-labeled IgE antibody and each antigen test sample were incubated at 371: 20 for 20 minutes. Thereafter, the fluorescence intensity of the antigen test sample-labeled antibody complex eluted with the malonate buffer used in Example 1 (3) was added to the ion exchange column used in Example 1 (3) and measured. : The control test (blank) was performed using phosphate buffered saline instead of the antigen test sample. Figure 6 shows the results. No increase in fluorescence intensity was observed for BSA, IgG, and HSA, whereas an increase was observed for DNP-BSA. Therefore, it was shown that the method of the present invention enables selective measurement of allergen.
実施例 3 ：定量性 Example 3: Quantitative
本発明方法の定量性及び定量範囲を調べるために、 モデルアレルゲンである D NP— B S A濃度が Om gZm し 0. 6 m g/m 1 , 1. 0 m g/m 1 , 1. 3 mg/m U 2. Om gZm し 又は 5. 0 m gZm 1である試料液 〔 0. 0 5Mマロン酸緩衝液 （p H 5) 中〕 を準備した。 この DN P— B S A溶液 1 0 0 / l と、 前記実施例 1 ( 1 ) で用いた 1. 8 n gZ 1の F I TC標識 I g E抗 体液 1 0 0 / 1 とを混合し、 3 7でで 2 0分間インキュベー ト した。 この各反応 液を、 前記実施例 1 ( 3 ) と同様の操作によって処理し、 溶出されるアレルゲン 一標識抗体複合体の蛍光強度を測定した。 その結果を図 7に示す。 アレルゲン^ 度が 0〜 2 m g/m 1の範囲において、 直線的な相関関係が示された。  In order to examine the quantification and quantification range of the method of the present invention, the concentration of D NP- BSA, which is a model allergen, was 0.6 mg / m 1, 1.0 mg / m 1, 1.3 mg / m U 2. A sample solution [0.05M malonic acid buffer (pH 5)] that was OmgZm or 5.0mgZm1 was prepared. This DNP-BSA solution (100/1) was mixed with 1.8 ng Z1 of FITC-labeled IgE antibody solution (100/1) used in Example 1 (1) above. And incubated for 20 minutes. Each reaction solution was treated in the same manner as in Example 1 (3), and the fluorescence intensity of the eluted allergen-labeled antibody complex was measured. Figure 7 shows the results. When the allergen was in the range of 0 to 2 mg / m1, a linear correlation was shown.
実施例 4 ： アルカリホスファタ一ゼ標識 I g E抗体による小麦アレルゲンの分析Example 4: Analysis of wheat allergen by alkaline phosphatase-transfer agent Ichize labeled I g E antibodies
( 1 ) 小麦アレルゲンの抽出液の調製 (1) Preparation of wheat allergen extract
薄力粉 1 0 gをジェチルェ一テル 1 0 0 m 1で洗浄した後、 2 0 0 0 Gで 3分 間、 遠心分離した。 この操作を 3回橾り返した。 沈澱物全量に 0. 1 Mリン酸緩 衝生理食塩水 （p H 7. 4 ) 1 0 0m l を加え、 2時間攪拌した。 上清を 0. 0 5 Mリン酸緩衝生理食塩水 （ p H 7. 4 ) 3 リ ッ トルに衬して 4 °Cで 7 2時間透 析し、 小麦アレルゲン抽出液 （3 5 ," g/m 1 ) とした：  After washing 100 g of the flour with 100 ml of Jetil ether, the mixture was centrifuged at 2000 G for 3 minutes. This operation was repeated three times. To the total amount of the precipitate, 100 ml of 0.1 M phosphate-buffered saline (pH 7.4) was added, followed by stirring for 2 hours. The supernatant was added to 3 liters of 0.05 M phosphate buffered saline (pH 7.4), and analyzed at 4 ° C for 72 hours to obtain a wheat allergen extract (35, "g). / m 1):
( 2 ) アルカリホスファターゼ標識 I g E抗体の調製  (2) Preparation of alkaline phosphatase-labeled IgE antibody
常法に従い、 実施例 4 ( 1 ) で調製した小麦アレルゲン抽出液で免疫したラッ トから抗小麦ァレルゲン抗血清 2 m 1を得た。 この抗血清に、 1 8重量 Z容量0 /。 になるように硫酸ナト リウムを加え、 得られた沈殿物を 0. 0 1Mリン酸緩衝液 (p H 8. 0) 2リ ッ トル中で 48時間透析を行った： その後、 DEAE弱塩基 性陰イオン交換カラム （5m l ; H i t r a p c o l umn ; フアルマシア製） に添加し、 0. 1 5M塩化ナト リウムを含有するリン酸緩衝液 （pH 8) を用い て、 0. 01M〜0. 2Mリン酸の範囲で直線濃度勾配をかけて溶出を行い、 抗 小麦アレルゲン一 I gE抗体画分を分取した。 According to a conventional method, a rat immunized with the wheat allergen extract prepared in Example 4 (1) was used. An anti-wheat allergen antiserum (2 ml) was obtained from the yeast. To this antiserum, 18 weight Z capacity 0 /. The resulting precipitate was dialyzed for 48 hours in 2 liters of 0.01 M phosphate buffer (pH 8.0) for 48 hours: Then, the DEAE weakly basic The solution is added to an anion exchange column (5 ml; Itrapcolumn; manufactured by Pharmacia), and the phosphate buffer (pH 8) containing 0.15 M sodium chloride is used to prepare 0.01 M to 0.2 M phosphate. Elution was performed by applying a linear concentration gradient in the range described above, and an anti-wheat allergen-IgE antibody fraction was collected.
得られた抗小麦アレルゲン一 I gE抗体画分を、 以下の方法でアルカルホスファ 夕一ゼにより標識した。  The obtained anti-wheat allergen-IgE antibody fraction was labeled with alkaline phosphatase by the following method.
アルカリホスファタ一ゼ （S I GMA社） 0. 4 5mgを、 0. I mM— Mg C 12 と 0. I mM— Z nC l 2 とを含む 0. 1Mトリス一塩酸緩衝液 （ p H 7. 0 ) 1 m lに溶解した。 この溶液 1 m 1に、 架橋剤 . N—サクシンィ ミジル一 3 - (2—ピリジルジチォ） プロピネー ト （以下、 S P D Pと称する ； フアルマシ ァ社製） 0. 5mgを添加し、 室温で 2時間インキュベートした後、 脱塩カラム を用いて未反応の S PDPを除去し、 S P DP結合アル力リホスファターゼを得 た。 これとは別にジチオスレイ ト一ル （DTT) により ヒンジ部ジスルフイ ド結 合を還元処理した抗小麦アレルゲン— I g E抗体画分 （ 1. l mgZm l ) 1 m 1 と、 前記 S PDP結合アルカリホスファタ一ゼ l m 1 とを混合し、 4°Cで 1 2 時間ィンキュベ一 トし、 アルカリホスファタ一ゼのァミノ¾と抗小麦アレルゲンAlkaline phosphatase Fatah Ichize (SI GMA Inc.) 0. 4 5mg, 0. I mM- Mg C 1 2 and 0. I mM- Z nC l 2 and 0. 1M Tris monohydrochloride buffer containing (p H 7 0) Dissolved in 1 ml. To 1 ml of this solution, 0.5 mg of a cross-linking agent .N-succinimidyl-1- (2-pyridyldithio) propionate (hereinafter referred to as SPDP; manufactured by Pharmacia) was added, and the mixture was incubated at room temperature for 2 hours. Then, unreacted SPDP was removed using a desalting column to obtain SPDP-bound allelic phosphatase. Separately, 1 ml of an anti-wheat allergen-IgE antibody fraction (1.1 mgZml) obtained by reducing the disulfide bond in the hinge region with dithiothreitol (DTT), After mixing with fat lm 1 and incubating at 4 ° C for 12 hours, the amino acid of alkaline phosphatase and anti-wheat allergen
— I gE抗体のチオール基との間を S PDPで架橋した。 得られた反 IS液をゲル 濾過カラムに添加し、 アルカリホスファターゼ標識抗小麦アレルゲン一 I gE抗 体と、 未反応の I g E抗体又は未反応の S P D P結合アル力リホスファターゼと を分離し、 アルカリホスファタ一ゼ標識抗小麦アレルゲン— I gE抗体画分 （7 μ g/m 1 ) を得た。 — Cross-linked with the thiol group of the IgE antibody with SPDP. The obtained anti-IS solution is added to a gel filtration column, and an alkaline phosphatase-labeled anti-wheat allergen-IgE antibody is separated from unreacted IgE antibody or unreacted SPDP-binding al lipophosphatase. A phosphatase-labeled anti-wheat allergen-IgE antibody fraction (7 μg / m 1) was obtained.
(3 ) 試料との接触  (3) Contact with sample
実施例 4 ( 2 ) で調製したアルカリホスファタ一ゼ標識抗小麦ァレルゲン一 I gE^t体 50 1 と、 実施例 4 ( 1！ で調製した麦アレルゲン抽出液 （35 μ g /m l ) 50," 1 とを混合し、 37°Cで 20分間イ ンキュベー ト し、 抗原抗体反 ίΐ；'を行った (4) イオン交換クロマトグラフィーによるアレルゲン一標識抗体複合体の分画 抗原抗体反応の終了後、 この混合液 1 00," 1を、 実施例 1 (3) で用いた陽 イオン交換カラムに添加した。 移動相としては、 50mMマロン酸緩衝液 （pH 5. 0) を用いて流速 1 m 1 /m i nで 2分間溶出した後、 続いて、 0〜0. 5 Mの直線濃度勾配の N a C 1水溶液を通液した。 溶出液は、 0. 25m lずつフ ラクシヨンとして分画した。 The alkaline phosphatase-labeled anti-wheat allergen-IgE ^ t form 501 prepared in Example 4 (2) and the wheat allergen extract (35 μg / ml) 50, prepared in Example 4 (1!) 1), incubated at 37 ° C for 20 minutes, and subjected to antigen-antibody reaction. (4) Fractionation of Allergen-Labeled Antibody Complex by Ion Exchange Chromatography After completion of the antigen-antibody reaction, this mixture 100, "1" was added to the cation exchange column used in Example 1 (3). The mobile phase was eluted with a 50 mM malonate buffer (pH 5.0) at a flow rate of 1 m1 / min for 2 minutes, followed by a linear concentration gradient of NaC from 0 to 0.5 M. The eluate was fractionated in 0.25 ml fractions as a fraction.
また、 分析には、 発光基質として 3 _ (2 ' —スピロアダマンタン） 一4—メ トキシ一 4一 (3" 一ホスホリロキシ） フエ二ルー 1， 2—ジォキセタン （AM PPD) 300 1を添力 Πし、 371Cで 15分間インキュベート した後、 ルミネッ センス （リーダ一） （BLR 301 ； A 1 0 k a杜） を用いて、 発光強度を測定 した。 その結果を図 8に示す。 図 8から明らかなように、 アレルゲン一標識抗体 複合体 （ピーク A) のみがマロン酸緩衝液溶出画分に溶出し、 0〜0. 5Mの直 線濃度勾配の N a C 1水溶液を通液したところ、 未反応のアルカリホスファタ一 ゼ標識抗小麦アレルゲン一 I gE抗体 （ピーク B) が溶出された。 すなわち、 抗 原抗体反応物と未反応のアルカリホスファタ一ゼ標識抗小麦アレルゲン— I g E 抗体とが、 よく分離されていた。 このように、 本発明方法によってアレルゲンを 分析することができた。  In addition, 3_ (2'-spiroadamantane) 14-methoxy-14- (3 "-phosphoryloxy) phenyl 1,2-dioxetane (AM PPD) 300 1 was used as a luminescent substrate in the analysis. After 15 minutes of incubation at 371C, the luminescence intensity was measured using Luminescence (Leader-1) (BLR 301; A10ka), and the results are shown in Figure 8. Then, only the allergen-labeled antibody complex (Peak A) eluted in the malonate buffer elution fraction and was passed through a NaCl aqueous solution with a linear concentration gradient of 0 to 0.5M. Alkaline phosphatase-labeled anti-wheat allergen-IgE antibody (Peak B) was eluted, that is, the unreacted alkaline phosphatase-labeled anti-wheat allergen-IgE antibody was unreacted. As described above, the present invention is well-separated. It was possible to analyze the allergen by.
実施例 5 ：定量性の検定 Example 5: Quantitative test
前記の実施例 4 (2) と同様の方法で既知量に調整した小麦アレルゲン及びァ ルカリホスファタ一ゼ標識抗小麦アレルゲン一 I g E抗体を用いて、 前記実施例 4 (3) 及び （4 ) の操作を行い、 定量性を検定した。 その結果を図 9に示す c なお、 図 9では、 カラムに試料全体を添加した場合の発光強度を 1 00%とした 場合の相対 ¾光強度を示す。  Using wheat allergen and alkaline phosphatase-labeled anti-wheat allergen-IgE antibody adjusted to a known amount in the same manner as in Example 4 (2) above, the procedure of Examples 4 (3) and (4) was repeated. The procedure was performed to test the quantitativeness. The results are shown in FIG. 9. c FIG. 9 shows the relative light intensity when the emission intensity when the entire sample was added to the column was 100%.
その結果、 小麦アレルゲン濃度が 0〜 60 μ g/m 1の範囲で直線関係が得ら れた。 このように、 本発明方法によって小麦アレルゲンを定量することができた： 実施例 6 ： アルカリホスファタ一ゼ標識 I g G抗体を用いたアレルゲン測定  As a result, a linear relationship was obtained when the wheat allergen concentration was in the range of 0 to 60 µg / m1. Thus, wheat allergens could be quantified by the method of the present invention: Example 6: Allergen measurement using alkaline phosphatase labeled IgG antibody
( 1 ) 抗,？一ラク トグロブリン一 I g G抗体を用いたアレルゲン測定  (1) Anti ,? Allergen measurement using mono-globulin-IgG antibody
常法に従い、 牛乳アレルゲンである つ一ラク トグロブリ ン （S I GMA杜；粉 末； 3回結晶標品） をラッ トに免疫し、 杭,？一ラク トグロプリン抗血清 2 m 1 を 得た。 得られた抗血清を、 プロテイン Aカラムに添加し、 I gG抗体画分を得た。 この I g G抗体画分を用いて、 前記実施例 4 (2) と同様の操作を行い、 アル力 リホスファタ一ゼ標識抗,？一ラク トグロブリン一 I g G抗体を得た。 In accordance with the usual method, the rat was immunized with a milk allergen, one-lactoglobulin (SI GMA forest; powder; three times crystal preparation), and stakes,? 1 ml of antiglobulin antiserum 2 ml Obtained. The obtained antiserum was added to a protein A column to obtain an IgG antibody fraction. Using the IgG antibody fraction, the same operation as in the above Example 4 (2) was carried out to obtain an anti-lipophosphatase-labeled anti- ,? One lactoglobulin-one IgG antibody was obtained.
得られたアル力リホスファタ一ゼ標識抗 ーラク トグロプリ ンー I gG抗体 (20 ^ g/m 1 ： 0. 05Mリ ン酸緩衝液中） 50 / 1 と , 3—ラク トグロプリ ン （30 gZm l : 0. 05Mリン酸緩衝液中） 50 1 とを室温で 20分間 ィンキュベ一 ト し、 抗原抗体反応を行った。  The resulting anti-lipophosphatase-labeled anti-lactoglobulin-IgG antibody (20 ^ g / m 1: in 0.05M phosphate buffer) 50/1 and 3-lactogloprin (30 gZm l: 0 (In a 0.05M phosphate buffer) was incubated at room temperature for 20 minutes to carry out an antigen-antibody reaction.
一方、 米、 小麦、 裔麦、 及び大豆を、 それぞれ、 粉砕し、 粉砕物に 3%塩化ナ トリウム水溶液を加えてホモジナイズした後、 遠心分離し、 更に遠心透析により 塩化ナトリウムを除いた抽出液を調製した。 また、 卵白を、 等量の水で希釈した 希釈液を調製した。 それぞれの抽出液又は希釈液 50 μ 1 (いずれも 30 gタ ンパク質/ m l ) と、 前記アルカリホスファタ一ゼ標識抗,？一ラク トグロブリ ン - I g G抗体 （20 gZm l ) 50 1とを室温で 20分間ィンキュベートし、 抗原抗体反応を行った。  On the other hand, rice, wheat, descendant wheat, and soybeans are each ground, and the ground material is homogenized by adding a 3% aqueous sodium chloride solution, and then centrifuged. Prepared. In addition, a diluted solution was prepared by diluting egg white with an equal amount of water. 50 μl of each extract or diluent (30 g protein / ml each) and the alkaline phosphatase-labeled anti- ,? One lactglobulin-IgG antibody (20 gZml) 501 was incubated at room temperature for 20 minutes to carry out an antigen-antibody reaction.
これらの各反応液 1 0 0 1 を、 前記実施例 4 (4 ) に記載の方法に従って、 分画し、 発光強度を測定した。 その結果を図 1 0に示す。 図 1 0においては、 被 検試料として,？一ラク トグロブリン溶液 （牛乳） を使用した場合の結果を 「令」 で示し、 卵白希釈液を使用した場合の結果を 「讕」 で、 小麦抽出液を使用した場 合の結果を 「A」 で、 裔麦抽出液を使用した場合の結果を 「X」 で、 大豆抽出液 を使用した場合の結果を 「*」 で、 米抽出液を使用した場合の結果を 「一」 で、 それぞれ示す c Each of these reaction solutions 1001 was fractionated according to the method described in Example 4 (4), and the luminescence intensity was measured. The results are shown in FIG. In FIG. 10, as the test sample,? The results when using a lactoglobulin solution (milk) are indicated by “令”, the results when using an egg white dilution are indicated by “液”, and the results when using a wheat extract are indicated by “A”. The results using the descendant barley extract are shown as `` X '', the results using the soybean extract are shown as `` * '', and the results using the rice extract are shown as `` one ''. c
被検試料として，？一ラク トグロブリン溶液 （牛乳） を使用した場合には、 ァレ ルゲン （ —ラク トグロブリ ン） 一標識抗体複合体 （ピーク A ) のみがマロン酸 緩衝液溶出画分に溶出し、 0〜 0. 5 Mの直線濃度勾配の N a C 1水溶液を通液 したところ、 未反応の標識抗 ,？ーラク トグロブリン一 I g G抗体 （ピーク B ) が 溶出された。 また、 被検試料として、 卵白希釈液、 小麦抽出液、 幕麦抽出液、 大 豆抽出液、 又は米抽出液を使用した場合には、 未反じ、の標識抗,？ーラク トグロブ リ ン一 I gG抗体のみが溶出された： すなわち、 抗原抗体反 )ΐ.物と未反 標識抗 体とがよく分離され、 抗原抗体複合体のみを分析することができた。 ( 2 ) 抗卵アレルゲン一 I g G抗体を用いたアレルゲン測定 As a test sample,? When mono-lactoglobulin solution (milk) was used, only allergen (—lactoglobulin) -labeled antibody complex (peak A) eluted in the malonic acid buffer elution fraction, and was When a 5 M linear concentration gradient of NaC1 aqueous solution was passed, unreacted labeled anti- ,? IgG antibody (peak B) was eluted. In addition, when egg white diluent, wheat extract, barley extract, soybean extract, or rice extract was used as a test sample, unlabeled, unlabeled,? Only the lactoglobulin-IgG antibody was eluted: that is, the antigen-antibody was well separated from the non-anti-labeled antibody, and only the antigen-antibody complex could be analyzed. (2) Allergen measurement using anti-egg allergen-IgG antibody
^一ラク トグロブリンの代わりに、 実施例 6 ( 1 ) で調製した卵白希釈液を抗 原として使用し、 実施例 6 ( 1 ) に記載の方法に従って、 抗卵アレルゲン一 I g G抗体画分を調製し、 この I g G抗体画分を用いてアルカリホスファタ一ゼ標識 抗卵アレルゲン— I g G抗体を調製し、 実施例 6 ( 1 ) で調製した卵白希釈液、 β —ラク トグロブリ ン溶液、 小麦抽出液、 薷麦抽出液、 大豆抽出液、 又は米抽出 液と抗原抗体反応を実施し、 抗原抗体複合体の分析を行った。  ^ Instead of lactoglobulin, the egg white dilution prepared in Example 6 (1) was used as an antigen, and the anti-egg allergen-IgG antibody fraction was prepared according to the method described in Example 6 (1). Using the IgG antibody fraction, an alkaline phosphatase-labeled anti-egg allergen-IgG antibody was prepared. The egg white dilution prepared in Example 6 (1), β-lactoglobulin The antigen-antibody reaction was performed with the solution, wheat extract, oat extract, soybean extract, or rice extract, and the antigen-antibody complex was analyzed.
結果を図 1 1に示す。 図 1 1においては、 被検試料として卵白希釈液を使用し た場合の結果を 「♦」 で、 ，3—ラク トグロブリン溶液 （牛乳） を使用した場合の 結果を 「謂」 で、 小麦抽出液を使用した場合の結果を 「Α」 で、 騫麦抽出液を使 用した場合の結果を 「X」 で、 大豆抽出液を使用した場合の結果を 「*」 で、 米 抽出液を使用した場合の結果を 「―」 で、 それぞれ示す。 抗卵アレルゲン— I g Gを用いた場合にも、 卵アレルゲン—標識抗体複合体と未反応標識抗体とがよく 分離され、 アレルゲン一標識抗体複合体を分析することができた。 また、 被検試 料として、 ，3—ラク トグロブリ ン溶液 （牛乳） 、 小麦抽出液、 騫麦抽出液、 大豆 抽出液、 又は米抽出液を使用した場合には、 未反応の標識抗卵アレルゲン一 I g G抗体のみが溶出された。  The results are shown in FIG. In Fig. 11, the results obtained when using the egg white diluent as the test sample are indicated by “♦”, and the results obtained when a 3,3-lactoglobulin solution (milk) is used are indicated by “so-called”. Use 場合 for the result when using the liquid, X for the result using the bean extract, * for the result using the soybean extract, and use the rice extract when using the soybean extract The result of the above is indicated by “-”. Even when the anti-egg allergen-IgG was used, the egg allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed. When a 3,3-lactoglobulin solution (milk), a wheat extract, a wheat extract, a soybean extract, or a rice extract is used as the test sample, unreacted labeled anti-egg allergen is used. Only one IgG antibody was eluted.
( 3 ) 抗米アレルゲン一 I g G抗体を用いたアレルゲン測定  (3) Allergen measurement using anti-rice allergen-IgG antibody
？一ラク トグロブリンの代わりに、 実施例 6 ( 1 ) で調製した米抽出液を抗原 として使用し、 実施例 6 ( 1 ) に記載の方法に従って、 抗米アレルゲン一 I g G 抗体画分を調製し、 この I g G抗体画分を用いてアルカリホスファタ一ゼ標識抗 米アレルゲン— I g G抗体を調製し、 実施例 6 ( 1 ) で調製した卵白希釈液、 ，3 ーラク トグロブリン溶液、 小麦抽出液、 裔麦抽出液、 大豆抽出液、 又は米抽出液 と抗原抗体反応を実施し、 抗原抗体複合体の分析を行った：  ? Using the rice extract prepared in Example 6 (1) as an antigen instead of monolactoglobulin, an anti-rice allergen-IgG antibody fraction was prepared according to the method described in Example 6 (1). Using the IgG antibody fraction, an alkaline phosphatase-labeled anti-rice allergen-IgG antibody was prepared, and the egg white diluent prepared in Example 6 (1), a 3,3-lactoglobulin solution, An antigen-antibody reaction was performed with wheat extract, descendant barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex:
結果を図 1 2に示す。 図 1 2においては、 被検試料として米抽出液を使用した 場合の結果を 「令」 で、 —ラク トグロブリ ン溶液 （牛乳） を使用した場合の結 果を 「驪」 で、 卵白希釈液を使用した場合の結果を 「A」 で、 小麦抽出液を使用 した場合の結果を 「X」 で、 裔麦抽出液を使用した場合の結果を 「*」 で、 大豆 抽出液を使用した場合の結果を 「一」 で、 それぞれ示す： 抗米アレルゲン一 I g Gを用いた場合にも、 米アレルゲン一標識抗体複合体と未反応標識抗体とがよく 分離され、 アレルゲン一標識抗体複合体を分析することができた。 また、 被検試 料として、 ーラク トグロブリン溶液 （牛乳） 、 卵白希釈液、 小麦抽出液、 薷麦 抽出液、 又は大豆抽出液を使用した場合には、 未反応の標識抗米アレルゲン一 I g G抗体のみが溶出された。 The results are shown in FIG. In Fig. 12, the results when rice extract was used as the test sample were indicated by “No.”, the results when a lactoglobulin solution (milk) was used were indicated by “Y”, and the egg white diluent was used. `` A '' indicates the result when used, `` X '' indicates the result when wheat extract was used, `` * '' indicates the result when wheat extract was used, and `` A '' when soybean extract was used. The results are shown as “one”, respectively: Anti-rice allergen – Ig Even when G was used, the rice allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed. In addition, when a test sample was used as a test sample, a lactoglobulin solution (milk), an egg white diluent, a wheat extract, a barley extract, or a soybean extract was used. Only G antibody was eluted.
( 4 ) 抗小麦アレルゲン一 I g G抗体を用いたアレルゲン測定  (4) Allergen measurement using anti-wheat allergen-IgG antibody
/3一ラク トグロブリンの代わりに、 実施例 6 ( 1 ) で調製した小麦抽出液を抗 原として使用し、 実施例 6 ( 1 ) に記載の方法に従って、 抗小麦アレルゲン一 I g G抗体画分を調製し、 この I g G抗体画分を用いてアル力リホスファタ一ゼ標 識抗小麦アレルゲン一 I g G抗体を調製し、 実施例 6 ( 1 ) で調製した卵白希釈 液、 ，3—ラク トグロブリ ン溶液、 小麦抽出液、 薷麦抽出液、 大豆抽出液、 又は米 抽出液と抗原抗体反応を実施し、 抗原抗体複合体の分析を行った。 / 3 Instead of lactoglobulin, the wheat extract prepared in Example 6 (1) was used as an antigen, and the anti-wheat allergen-IgG antibody fraction was used according to the method described in Example 6 (1). The IgG white fraction was used to prepare an anti-wheat allergen-IgG antibody, which was used as an anti-wheat allergen, using the IgG antibody fraction. The egg white diluent prepared in Example 6 (1), An antigen-antibody reaction was performed with a lactoglobulin solution, a wheat extract, a barley extract, a soybean extract, or a rice extract, and the antigen-antibody complex was analyzed.
結果を図 1 3に示す。 図 1 3においては、 被検試料として小麦抽出液を使用し た場合の結果を 「♦」 で、 ーラク トグロブリン溶液 （牛乳） を使用した場合の 結果を 「謹」 で、 卵白希釈液を使用した場合の結果を 「A」 で、 裔麦抽出液を使 用した場合の結果を 「X」 で、 大豆抽出液を使用した場合の結果を 「*」 で、 米 抽出液を使用した場合の結果を 「一」 で、 それぞれ示す。 抗小麦アレルゲン一 I g Gを用いた場合にも、 小麦アレルゲン一標識抗体複合体と未反応標識抗体とが よく分離され、 アレルゲン一標識抗体複合体を分析することができた また、 被 検試料として、 ーラク トグロブリ ン溶液 （牛乳） 、 卵白希釈液、 裔麦抽出液、 大豆抽出液、 又は米抽出液を使用した場合には、 未反応の標識抗小麦アレルゲン - I g G抗体のみが溶出された。  The results are shown in FIG. In Fig. 13, the results when wheat extract was used as the test sample are indicated by “♦”, the results when the lactoglobulin solution (milk) was used are indicated by “K”, and the egg white diluent was used. `` A '', `` X '' indicates the result of using wheat seed extract, `` * '' indicates the result of using soybean extract, and `` * '' indicates the result of using rice extract. The results are indicated by "one". Even when anti-wheat allergen-IgG was used, the wheat allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed. When using lactoglobulin solution (milk), egg white diluent, descendant barley extract, soybean extract, or rice extract, only unreacted labeled anti-wheat allergen-IgG antibody is eluted. Was.
( 5 ) 抗裔麦アレルゲン一 I g G抗体を用いたアレルゲン測定  (5) Allergen measurement using descendant wheat allergen-IgG antibody
ーラク トグロブリ ンの代わりに、 実施例 6 ( 1 ) で調製した裔麦抽出液を抗 原として使用し、 実施例 6 ( 1 ) に記載の方法に従って、 抗裔麦アレルゲン一 I g G抗体画分を調製し、 この I g G抗体画分を用いてアルカリホスファターゼ標 識抗騫麦アレルゲン一 I g G抗体を調製し、 実施例 6 ( 1 ) で調製した卵白希釈 液、 ーラク トグロブリン溶液、 小麦抽出液、 騫麦抽出液、 大豆抽出液、 又は米 抽出液と抗原抗体反応を実施し、 抗原抗体複合体の分析を行った 結果を図 1 4に示す。 図 1 4においては、 被検試料として薷麦抽出液を使用し た場合の結果を 「♦」 で、 ^—ラク トグロブリン溶液 （牛乳） を使用した場合の 結果を 「固」 で、 卵白希釈液を使用した場合の結果を 「A」 で、 小麦抽出液を使 用した場合の結果を 「X」 で、 大豆抽出液を使用した場合の結果を 「*」 で、 米 抽出液を使用した場合の結果を 「一」 で、 それぞれ示す。 抗騫麦アレルゲン一 I g Gを用いた場合にも、 騫麦アレルゲン—標識抗体複合体と未反応標識抗体とが よく分離され、 アレルゲン一標識抗体複合体を分析することができた。 また、 被 検試料として、 ーラク トグロブリ ン溶液 （牛乳） 、 卵白希釈液、 小麦抽出液、 大豆抽出液、 又は米抽出液を使用した場合には、 未反応の標識抗薷麦アレルゲン - I g G抗体のみが溶出された。 In accordance with the method described in Example 6 (1), the descendant wheat allergen-IgG antibody fraction was used according to the method described in Example 6 (1), using the descendant wheat extract prepared in Example 6 (1) as an antigen instead of lactoglobulin. Using the IgG antibody fraction, an alkaline phosphatase-labeled anti-allergen-IgG antibody was prepared, and the egg white diluent, lactoglobulin solution, and wheat prepared in Example 6 (1). An antigen-antibody reaction was performed with the extract, barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex. The results are shown in FIG. In Fig. 14, egg white dilution is shown as “♦” when the barley extract was used as the test sample, and as “solid” when the ^ -lactoglobulin solution (milk) was used as the test sample. `` A '' indicates the result when using the soybean extract, `` X '' indicates the result when using the wheat extract, `` * '' indicates the result when using the soybean extract, and the rice extract was used. The result in each case is indicated by “one”. In the case of using the anti-allergen-allergen-IgG, the allergen-allergen-labeled antibody conjugate was well separated from the unreacted labeled antibody, and the allergen-labeled antibody conjugate could be analyzed. In addition, when the test sample used was a lactoglobulin solution (milk), egg white diluent, wheat extract, soy extract, or rice extract, unreacted labeled anti-barley allergen-IgG Only the antibody was eluted.
( 6 ) 抗大豆アレルゲン一 I g G抗体を用いたアレルゲン測定  (6) Allergen measurement using anti-soy allergen-IgG antibody
^一ラク トグロブリンの代わりに、 実施例 6 ( 1 ) で調製した大豆抽出液を抗 原として使用し、 実施例 6 ( 1 ) に記載の方法に従って、 抗大豆アレルゲン一 I g G抗体画分を調製し、 この I g G抗体画分を用いてアルカリホスファタ一ゼ標 識抗大豆アレルゲン一 I g G抗体を調製し、 実施例 6 ( 1 ) で調製した卵白希釈 液、 ーラク トグロブリ ン溶液、 小麦抽出液、 薷麦抽出液、 大豆抽出液、 又は米 抽出液と抗原抗体反応を実施し、 抗原抗体複合体の分析を行つた。  ^ The soybean extract prepared in Example 6 (1) was used as an antigen instead of lactoglobulin, and the anti-soy allergen-IgG antibody fraction was used according to the method described in Example 6 (1). Using the IgG antibody fraction, alkaline phosphatase-labeled anti-soy allergen-IgG antibody was prepared, and the egg white diluent and lactoglobulin solution prepared in Example 6 (1). An antigen-antibody reaction was performed with wheat extract, barley extract, soybean extract, or rice extract to analyze the antigen-antibody complex.
結果を図 1 5に示す。 図 1 5においては、 被検試料として大豆抽出液を使用し た場合の結果を 「♦」 で、 ，3—ラク トグロブリ ン溶液 （牛乳） を使用した場合の 結果を 「園」 で、 卵白希釈液を使用した場合の結果を 「A」 で、 小麦抽出液抽出 液を使用した場合の結果を 「X」 で、 騫麦抽出液を使用した場合の結果を 「*」 で、 米を使用した場合の結果を 「―」 で、 それぞれ示す。 抗大豆アレルゲン— I g Gを用いた場合にも、 大豆アレルゲン一標識抗体複合体と未反応標識抗体とが よく分離され、 アレルゲン一標識抗体複合体を分析することができた。 また、 被 検試料として、 ーラク トグロブリ ン溶液 （牛乳） 、 卵 A希釈液、 小麦抽出液、 薔麦抽出液、 又は米抽出液を使用した場合には、 未反応の標識抗大豆アレルゲン ― I G抗体のみが溶出された c The results are shown in FIG. In Fig. 15, the results when soybean extract was used as the test sample are indicated by “♦”, and the results when 3,3-lactoglobulin solution (milk) was used are indicated by “garden”. `` A '' indicates the result when using the extract, `` X '' indicates the result when the wheat extract was used, `` * '' indicates the result when the wheat extract was used, and rice was used. The results in each case are indicated by “-”. Even when the anti-soy allergen-IgG was used, the soy allergen-labeled antibody complex and the unreacted labeled antibody were well separated, and the allergen-labeled antibody complex could be analyzed. In addition, when the test sample used was a lactoglobulin solution (milk), an egg A dilution, a wheat extract, a rose extract, or a rice extract, unreacted labeled anti-soy allergen-IG antibody Only eluted c
実施例 7 ：定量性の検定 Example 7: Quantitative test
前記の実施例 6 ( 2 ) で調製した既知量の,？一ラク トグロブリンを含む溶液、 及びアルカリホスファタ一ゼ標識抗,？一ラク トグロプリ ンー I gG抗体を用いて、 前記実施例 4 (3) 及び （4) の操作を行い、 定量性を検定した。 その結果を図 1 6に示す。 なお、 図 1 6では、 カラムに添加した試料全体の発光強度を 1 00 %とした場合の相対発光強度を示す。 A known amount of?, Prepared in Example 6 (2) above. A solution containing monolactoglobulin, And alkaline phosphatase labeled anti- ,? Using the 1-lactoglobulin-IgG antibody, the operations of Examples 4 (3) and (4) were performed, and the quantitativeness was tested. Figure 16 shows the results. FIG. 16 shows the relative luminescence intensity when the luminescence intensity of the whole sample added to the column is 100%.
図 1 6に示すように、 一ラク トグロプリン濃度が 0〜 80 g/m 1の範囲 で直線関係が得られた。 このように、 本発明方法ではアレルゲンの定量が可能で あることが確認された。  As shown in FIG. 16, a linear relationship was obtained when the concentration of monolactoglobulin was in the range of 0 to 80 g / m 1. As described above, it was confirmed that the method of the present invention can quantify allergens.
実施例 8 ： ? 1丁(：標識1 gE抗体による食物アレルゲンの分析 Example 8: Analysis of food allergens by using 1 (: labeled 1 gE antibody
牛乳、 卵白、 米、 小麦、 薷麦、 又は大豆に対する各アレルギー患者の血清を用 いて、 前記実施例 4 (2 ) に記載の方法に従って、 それぞれの I g E抗体を得た。 得られた各アレルギー患者の I gE抗体を用いて、 実施例 1 ( 1 ) に記載の方法 に従って、 それぞれの F I TC標識 I gE抗体を得た。  Each IgE antibody was obtained according to the method described in Example 4 (2) above, using the serum of each allergy patient to milk, egg white, rice, wheat, oats, or soybean. Using the obtained IgE antibodies of allergic patients, respective FITC-labeled IgE antibodies were obtained according to the method described in Example 1 (1).
被検試料としては、 牛乳、 並びに実施例 6 ( 1 ) で調製した卵白希釈液、 小麦 抽出液、 裔麦抽出液、 大豆抽出液、 及び米抽出液を用いた。 各アレルギー患者の F I TC標識 I gE抗体 （ 1. 5 n g) と、 前記各被検試料 （アレルゲンタンパ ク質量で 2 m g) とを 37°Cで 20分間反応させ、 抗原抗体反応を行った。 各反 応液について、 実施例 1 (3) に記載の方法に従って、 アレルゲン一標識抗体複 合体由来の蛍光強度を測定した。  As test samples, milk, egg white diluent, wheat extract, descendant barley extract, soybean extract, and rice extract prepared in Example 6 (1) were used. The FTC-labeled IgE antibody (1.5 ng) of each allergic patient and each of the test samples (2 mg in terms of allergen protein mass) were allowed to react at 37 ° C for 20 minutes to perform an antigen-antibody reaction. For each reaction solution, the fluorescence intensity derived from the allergen-labeled antibody complex was measured according to the method described in Example 1 (3).
牛乳アレルギー患者由来の I g E抗体から調製した F I T C標識 I g E抗体を 用いた場合の結果を図 1 7に示す。 図 1 7に示す記号は、 図 1 0に示す記号と同 じ意味である。 図 1 7に示すように、 牛乳アレルギー患者由来の I gE抗体から 調製した F I TC標識 I g E抗体を用いた場合には、 牛乳に対してのみアレルゲ ンー標識抗体複合体由来の蛍光を分析することができ、 患者が牛乳に対してァレ ルギーをもっと判定することができた c Fig. 17 shows the results obtained using FITC-labeled IgE antibodies prepared from IgE antibodies derived from milk allergy patients. The symbols shown in FIG. 17 have the same meanings as the symbols shown in FIG. As shown in Figure 17, when FITC-labeled IgE antibodies prepared from IgE antibodies from milk allergy patients are used, the fluorescence from the allergen-labeled antibody complex is analyzed only for milk. it can, c the patient was able to more determined § Les Energy with respect to milk
同様に、 卵、 米、 小麦、 養麦、 又は大豆の各アレルギー患者由来の I gE抗体 から調製した F I TC標識 I gE抗体を用いた場合の結果を、 それぞれ、 図 1 S Similarly, the results obtained using FITC-labeled IgE antibodies prepared from IgE antibodies from allergic patients of egg, rice, wheat, wheat, or soybean are shown in Fig.
(卵ァレルギ—患者由来の I g E抗体使用） 、 図 1 9 (米ァレルギ一患者由来の I gE抗体使用） 、 図 20 (小麦アレルギー患者由来の I gE抗体使用） 、 図 2 1 (喬麦アレルギー患者由来の I g E抗体使用） 、 及び図 22 (大豆アレルギー 患者由来の I g E抗体使用） に示す。 図 1 8〜図 2 2に示す記号は、 それぞれ、 図 1 1〜図 1 5に示す記号と同じ意味である。 図 1 8〜図 2 2に示すように、 卵、 米、 小麦、 騫麦、 又は大豆に対する各アレルギー患者由来の I g E抗体から調製 した F I TC標識 I gE抗体を用いた場合には、 それぞれ、 卵、 米、 小麦、 騫麦、 又は大豆に対してのみ、 アレルゲン—標識抗体複合体由来の蛍光を分析すること ができ、 患者のアレルゲンを同定することができた。 (Egg allergy-using IgE antibody from patient), Fig. 19 (using IgE antibody from a single patient from rice allergy), Fig. 20 (using IgE antibody from wheat allergy patient), Fig. 21 (high quality wheat) Using IgE antibodies from allergic patients), and Fig. 22 (Soybean allergy) Using patient-derived IgE antibody). The symbols shown in FIGS. 18 to 22 have the same meanings as those shown in FIGS. 11 to 15, respectively. As shown in Figure 18 to Figure 22, when using FITC-labeled IgE antibodies prepared from IgE antibodies from allergic patients to egg, rice, wheat, wheat, or soybean, respectively, Only eggs, rice, wheat, wheat, or soy could be analyzed for fluorescence from the allergen-labeled antibody complex, and the patient's allergen could be identified.
実施例 9 ： カラムへの連続添加によるアレルゲンの分析 Example 9: Analysis of allergen by continuous loading on column
実施例 1 ( 1 ) で調製した 1. 8 n gZ 1の F I TC標識抗 DNP— I g E 抗体 1 0 0 / 1 と、 0. 6 mg/m l 、 1. 0 m g/m 1 , 1. 3 mg/m l 、 又は 2. O m gノ m lのジニトロフエ二ル化牛血清アルブミ ン （DN P— B SA) 1 0 0 1 とをそれぞれ混合し、 3 7 で 2 0分間ィンキュベートした。 このと き、 対照試験 （ブランク） として、 DNP— B S A水溶液の代わりに、 水を使用 して同様の操作を実施した。 これらの反応液 1 0 0 / 1 を、 5 0mMマロン酸緩 衝液 （p H 5. 0) で平衡化した、 実施例 1 ( 3 ) で用いた陽イオン交換カラム に、 2 0分毎に添加した。 なお、 試料を添加してから、 次の試料を添加するまで の間に、 カラムの塩水洗浄は一切行わなかった。 添加は、 DNP— B SAの各濃 度毎に 4回ずつ添加した。 移動相としては、 5 0 mMマロン酸緩衝液 （p H 5. 0 ) を流速 1 m 1 Zm i nで用いた。 溶出液は 0. 2 5 m lずつフラクションと して分画し、 溶出液の蛍光強度を蛍光分光光度計 （F— 1 2 0 0型、 [：]立製作所 製） で測定した。  FITC-labeled anti-DNP-IgE antibody of 1.8 ng z1 prepared in Example 1 (1) 100/1, 0.6 mg / ml, 1.0 mg / m1, 1. 3 mg / ml or 2. O mg of dinitrophenylated bovine serum albumin (DNP-BSA) 1001 was mixed with each other and incubated at 37 for 20 minutes. At this time, as a control test (blank), the same operation was performed using water instead of the DNP-BSA aqueous solution. The reaction mixture 100/1 was added every 20 minutes to the cation exchange column used in Example 1 (3), which had been equilibrated with 50 mM malonic acid buffer solution (pH 5.0). did. The column was not washed with salt water at all between the sample addition and the next sample addition. The addition was performed four times at each concentration of DNP-BSA. As a mobile phase, a 50 mM malonic acid buffer solution (pH 5.0) was used at a flow rate of 1 m 1 Zmin. The eluate was fractionated in 0.25 ml fractions, and the fluorescence intensity of the eluate was measured with a fluorescence spectrophotometer (F-1200, [:] manufactured by Tachi Seisakusho).
溶出された抗原抗体複合体由来の蛍光強度を図 2 3に示す。 図 2 3では、 各反 応液を^加した時点を 「†」 で示し、 2. Om g/m 1 の DN P— B S Aと混合 した反応液の添加を 「A」 で、 1. 3 m gZm 1の DNP— B S Aと混合した反 応液の 加を 「B」 で、 1. 0 m g /m 1の D N P— B S Aと混合した反応液の 添加を 「C」 で、 0. 6 m g/m 1の DN P— B S Aと混合した反応液の添加を 「D」 で、 水と混合した反応液の添加を 「E」 で示す。 いずれの濃度の DN P 一 B S Aにおいても、 それぞれ安定した値を示し、 カラムを塩水洗浄せずに試料 を連続添加しても、 使用するイオン交換樹脂の、 未反応の F I TC標識 I g E抗 体に対する吸着容量の範囲内では、 定量性があることが示された c 実施例 10 ：陰ィオン交換力ラムを用いた未反応抗体の分析 The fluorescence intensity derived from the eluted antigen-antibody complex is shown in FIG. In Fig. 23, the point at which each reaction solution was added is indicated by "†", and the addition of the reaction solution mixed with 2. Omg / m1 of DNP-BSA was indicated by "A", and the time of addition was 1.3 m. The addition of the reaction mixture mixed with gZm1 DNP-BSA was performed with “B”, and the addition of the reaction mixture mixed with 1.0 mg / m1 DNP-BSA was added with “C” at 0.6 mg / m The addition of the reaction mixture mixed with 1 DNP-BSA is indicated by “D”, and the addition of the reaction mixture mixed with water is indicated by “E”. Regardless of the concentration of DNP-BSA at any concentration, each value shows a stable value, and even if the sample is continuously added without washing the column with salt water, the unreacted FITC-labeled IgE Within the range of the adsorption capacity for the body, it was shown to be quantitative c Example 10: Analysis of unreacted antibody using anion exchange force ram
実施例 1 (1) と同様の操作を行い、 抗 DNP— I g E抗体を F I TC標識し た。 続いて、 実施例 1 (2) と同様の操作を行い、 F I TC標識抗 DNP— I g E抗体 （1. S n gZ^ l ) 100/ lと DNP— BSA (モデルアレルゲン） The same operation as in Example 1 (1) was performed, and the anti-DNP-IgE antibody was labeled with FITC. Subsequently, the same operation as in Example 1 (2) was performed, and FITC-labeled anti-DNP-IgE antibody (1. SngZ ^ l) 100 / l and DNP-BSA (model allergen)
(2 mg/m 1) 100 / 1を室温で 20分間反) ΐ、させた。 (2 mg / m 1) 100/1 was counteracted at room temperature for 20 minutes.
この反応液を、 予め 5 OmMグリシン—塩化ナトリゥム一水酸化ナト リ ウム緩 衝液 （pH 9) で平衡化した陰イオン交換カラム （H I LOAD Q S e p h a r o s e Fa s t F l ow ;直径 = 1 6 X 25 mm； ファルマシア製） に 添加した。 移動相としては、 平衡化に用いた前記緩衝液 （pH9. 0) を用いて 流速 1 m 1 /m i nで 2分 30秒間溶出した後、 続いて、 0〜 0. 5 Mの直線濃 度勾配一 N a C 1水溶液を通液した。 溶出液は 0. 25m lずつフラクショ ンと して分画した。 溶液の蛍光強度を蛍光分光光度計 （F— 1200型、 日立製作所 製） で測定した。 結果を図 24に示す。 図 24に示すように、 抗原抗体複合体 An anion exchange column (HI LOAD QS epharose Fast Flow; diameter = 16 x 25 mm) previously equilibrated with 5 OmM glycine-sodium chloride monohydroxide buffer (pH 9) was used. Pharmacia). The mobile phase was eluted with the buffer (pH 9.0) used for equilibration at a flow rate of 1 m1 / min for 2 minutes and 30 seconds, followed by a linear concentration gradient of 0 to 0.5 M. One NaC1 aqueous solution was passed. The eluate was fractionated in 0.25 ml fractions. The fluorescence intensity of the solution was measured with a fluorescence spectrophotometer (F-1200, manufactured by Hitachi, Ltd.). The results are shown in FIG. As shown in Figure 24, the antigen-antibody complex
(ピーク A) のみが、 5 OmMグリシン一塩化ナトリウム—水酸化ナトリウム緩 衝液溶出画分に溶出し、 0〜 0. 5 Mの直線濃度勾配の N a C 1水溶液を通液し たところ、 未反応の F I TC標識 I gE抗体 （ピーク B) が溶出された。 すなわ ち、 陽イオン交換体の代わりに陰イオン交換体を用いても、 抗原抗体反応物と未 ¾Dの標識抗原とがよく分離され、 抗原抗体複合体のみを分析することができた： 産業上の利用可能性 Only (Peak A) eluted in the eluted fraction of 5 OmM glycine sodium monochloride-sodium hydroxide buffer solution, and passed through a NaCl aqueous solution with a linear concentration gradient of 0 to 0.5 M. The FITC-labeled IgE antibody (peak B) of the reaction was eluted. In other words, even when an anion exchanger was used instead of a cation exchanger, the antigen-antibody reactant and the unlabeled antigen were well separated, and only the antigen-antibody complex could be analyzed. Availability on
本発明により、 従来にない簡便な操作で、 迅速にアレルゲンを定量することが できる c According to the present invention, allergens can be quickly quantified by a simple operation that has never been performed before.c
以上、 本発明を特定の実施態様に沿って説明したが、 当業者に自明の変形は本 発明の範囲に含まれる c  Although the present invention has been described with reference to specific embodiments, modifications obvious to those skilled in the art are included in the scope of the present invention.

Claims

求 の 範 囲 Range of request
1. ( 1 ) 検査対象アレルゲンを含有する疑いのある試料と、 前記の検査対象ァ レルゲンに特異的な標識抗体とを、 前記の検査対象アレルゲンと前記標識抗体と の抗原抗体反応が可能な条件下で接触させる工程、 （2) 得られた反応液からィ オン交換ク口マトグラフィ一により、 検査対象アレルゲンに未反応の標識抗体を 除去する工程、 そして （3) 溶出される検査対象アレルゲン一標識抗体複合体由 来の信号を検出する工程を含むことを特徴とする、 アレルゲンの分析方法。 1. (1) A sample suspected of containing the test allergen and a labeled antibody specific to the test allergen are subjected to conditions that allow an antigen-antibody reaction between the test allergen and the labeled antibody. (2) removing the unreacted labeled antibody from the obtained reaction solution by ion exchange chromatography, and (3) eluted test allergen label A method for analyzing an allergen, comprising a step of detecting a signal derived from an antibody complex.
2. 標識が、 蛍光性化合物、 酵素、 又は放射性物質である、 請求項 1に記載の方 法。  2. The method according to claim 1, wherein the label is a fluorescent compound, an enzyme, or a radioactive substance.
3. 蛍光性化合物としてフルォレセインイソチオシァネー トを用いる請求項 2に 記載の方法。  3. The method according to claim 2, wherein fluorescein isothiosinate is used as the fluorescent compound.
4. 酵素として、 アルカリホスファタ一ゼ、 3 _D_ガラク トシダ一ゼ、 西洋ヮ サビペルォキシダ一ゼ、 又はカテコール一 0 —メチルトランスフェラ一ゼを用い る請求項 2に記載の方法。  4. The method according to claim 2, wherein alkaline phosphatase, 3_D_galactosidase, horseradish siperoperoxidase, or catechol 10-methyltransferase is used as the enzyme.
5. イオン交換クロマトグラフィーと して、 陽イオン交換クロマトグラフィーを 用いる請求項 1に記載の方法。  5. The method according to claim 1, wherein cation exchange chromatography is used as the ion exchange chromatography.
6. 未反応標識抗体を陽イオン交換体に吸着させ、 検査対象アレルゲン一標識抗 体複合体を酸性緩衝液で溶出させる、 請求項 5に記載の方法。  6. The method according to claim 5, wherein the unreacted labeled antibody is adsorbed on a cation exchanger, and the test allergen-labeled antibody complex is eluted with an acidic buffer.
7. 酸性緩衝液が、 p H 4. 5〜 6である、 請求項 6に記載の方法。  7. The method of claim 6, wherein the acidic buffer has a pH of 4.5-6.
8. イオン交換クロマトグラフィーとして、 陰イオン交換クロマトグラフィーを 用いる請求項 1に記載の方法。  8. The method according to claim 1, wherein anion exchange chromatography is used as the ion exchange chromatography.
9. 抗体が I gE又は I g Gである、 請求項】〜 8のいずれか一項に記載の方法。  9. The method according to any one of claims 1 to 8, wherein the antibody is IgE or IgG.
1 0. 前記の工程 （ 1 ) 、 工程 （ 2 ) 及び工程 （ 3 ) を橾り返し連続的に行う、 請求項 1〜 8のいずれか一項に記載の分析方法 c 10. The analysis method c according to any one of claims 1 to 8, wherein the steps (1), (2) and (3) are repeatedly and continuously performed.
PCT/JP1996/002707 1995-09-22 1996-09-20 Method for the analysis of allergen WO1997011368A1 (en)

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